“I‘ v—— MENO ACID METABGLESM BY RUMEN MiCRQORGANISMS Thai: for the Dagm 0f Ph. D. MICHGAN NATE UNNERSETY Tran? R. Lewis 1951 This is to certify that the thesis entitled AMINO ACID METABOLISM BY RUMEN MICROORGANISMS presented by TRENT R. LEWIS has been accepted towards fulfillment of the requirements for Mdegree in_DAIRX__ W fl’t/ éjfliA/Z y Major professor ~ Date APRIL 14. 1961 0-169 LIBRARY Michigan State University AMINO ACID METABOLISM BY RUMEN MICROORGANISMS By (I Trent R1 Lewis A THESIS Submittedto Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Dairy 1961 “7/3 2/2 / ABSTRACT AMINO ACID METABOLISM BY RUMEN MICROORGANISES by Trent R. Lewis This investigation had a fourfold purpose. Cata- bolic reactions of amino acids were compared and quanti- tated in both in xitgg mediums--rumen liquor and washed suspensions of rumen microorganisms. Each amino acid medium was also examined chromatographically for inter- mediary products which could have pronounced physiological activity within the host. Certain materials were added to washed cell suspensions in an effort to increase the amino acid dissimilation rate. Lastly, Learginine, L-lysine and DL—tryptophan were studied in IEZQ to compare in ziggg and in XAXQ dissimilation patterns and the effect of amino acid administration through the rumen fistula on the plasma amino acids of jugular blood. Rumen fluid which was to serve as inocula and washed cell sources was collected at three to four hours after feeding. The amino acids were dissolved in each medium to give amino acid concentrations of 10 micromoles per ml. In the in 2139 studies, each amino acid was added two hours after feeding in quantities to give amino acid concentrations of approximately 20 micromoles per ml. Trent R. Lewis The post incubation mediums were examined chromatograph- ically and spectrOphotometrically for catabolic products. Plasma amino acids from jugular blood were examined quali- tatively by paper chromatOgraphy zero, one, two and four hours after the administration of the amino acid. Serine, aspartic acid, glutamic acid, arginine, lysine, cysteine, cystine, threonine and phenylalanine were readily dissimilated when added to both mediums (47 to 100% in rumen liquor; 21 to 99% in washed cells). TryptOphan, histidine, methionine, ornithine, valine, ala- nine, leucine, isoleucine, delta amino valeric acid, gly- cine, hydroxyproline and proline were dissimilated at lesser rates (8 to.37% in rumen liquor; l to 22% in washed cells). The dissimilation rates were more rapid and com- plete in rumen fluid studies than in washed cell suspen- sions. Three or four amino acids incubated together dif- fered from the summation of the ammonia formed from each amino acid only in the cases where proline and alanine were incubated together. The individual usage of 48 hour enriched cultures, pyridoxamine, pyridoxal phosphate, mag- nesium ions, all potassium buffers, methylene blue or catalase in washed cell incubations failed to significantbr promote ammonia production over the control values and were still low as compared to rumen liquor and ig gixg am- monia production. Ammonia production and amino acid Trent R. Lewis disappearance, as noted by paper chromatography, were closely correlated. The D- and L- forms of tryptOphan and serine were both dissimilated. Arginine yielded ornithine, putrescine"and delta amino valeric acid. Lysine yielded cadaverine and delta amino valeric acid whereas ornithine yielded delta amino valeric acid. Penicillin at 5 I.U./ml. did not inhibit any of the dissimilations whereas 50 I.U. caused a marked inhibition. Tests for amine production from casein hydrolyzate and in- dividual amino acids at pH 4.5, 5.5 and 6.5 were negative, except for cadaverine and putrescine. Arginine produced the highest levels of ammonia in eight hour rumen liquor incubations. The presence of arsenate or fluoride in- creased ammonia production from serine over the control values. Glutaric acid was not dissimilated in zitrg by rumen microorganisms. The in zigg dissimilations of L-arginine, L-lysine and DL-tryptoPhan were in good agreement with the in zitgg studies. Arginine and lysine both produced delta amino valeric acid. Arginine also yielded ornithine. Indole and skatole were formed from tryptOphan. Rumen ammonia levels in 1319 paralleled what would have been expected from the ig,zi§gg studies. The administration of readily dissimilatable amino acids to the rumen had a generally positive effect on the amino acids from jugular blood Trent R. Lewis drawn the first four hours following amino acid adminis- tration rather than specifically raising the level of the amino acid administered. ACKNOWLEDGEKENTS The author wishes to express his sincere gratitude to Dr. R. S. Emery for his guidance and help in the thesis problem and in the preparation of this manuscript and to Dr. J. W. Thomas for assistance in preparation of this manuscript. He is also grateful to Dr. C. F. Huffman and Dr. C. A. Lassiter for the roles they played during the course of this study. Their helpful criticisms and valu- able suggestions were major factors in this desire becom- ing a reality. The writer is especially indebted to his parents, Prince E. Lewis and kargaret L. Lewis, who made supreme ‘ sacrifices during the course of the author's education. One gave his life and the other her health. This manu- script is gratefully dedicated to these two individuals. Finally, I am unable to express adequately my gratefulness to Dr. J. C. Shaw and Mr. Malcolm Kerr, whose consideration and personal generosity twice made it possi— ble for me to remain in college. ii LABLL‘J OF COIiTILIi S ) INTRODUCTION . . . . . . . . . . . . . . . . . . REVIEJ OF LITERATURE . . . . . . . . . . ... . . Protein Catabolism. . . . . . . . . . . . Amino Acid Metabolism . . . . Glyc ine O O O O O C O O O O 0 O O O Alanine O O O O O O O O O O O O O O Serine -,- . . . . . . . . . . . . Threonine. . . . . . . . . . . . . Aspartic acid. . . . . . . . . . . Glutamic acid. . . . . . . . . . . Valine O O O 0 O O O O O O O O O O Leucine and isoleucine . . . . . . Phenylalanine. . . . . . . . . . . Tyrosine . . . . . . . . . . . . . HiStidine O O O O O O O O O O O O O Proline and hydroxyproli e . . . . TryptOphan . . . . . . . . . . . Cystine and cysteine . . . . . . . {ethionine O O O O O O O O O O O O LySine O O O O 0 O O O O O 0 O O O Arginine and ornithin ne . . . . . . The Stickland Reaction. . . . . . . . . . Non-Protein Nitrogen. . . . . . . . . . . Protein Anabolism . . . . . . . . . . . . EXPERID’- HIVTfl PROCL PDUE o o o o o o o o o o e o o In Vitro Studies. . . . . . . . . . . . . General methods. . . . . . . . . . Glutaric acid analysis . . . . . . Amino acid analysis. . . . . . . . Analysis of tryptOphan derivatives Amine analysis . . . . . . . . . . In Vivo Studies . . . . . . . . . . . . . RESULTS. 0 O O O O O O O O O O O O O O O 0 In Vitro Studi ies. . . . . . . . . . . . . iii 0 O O O O O O O O O O O O O O O O TABLE CF CCLTEYTS (Continued) Experiment 1. Use of Enriched Cultures. 76 Experiment 2. Pyridoxamine. . . . . . Experiment 5. Pyridoxal Phosphate . . . 78 EXperiment 4. Magnesium ion . . . . . . 79 Experiment 5. Potassium ion . . . . . 79 Experiment 6. Methylene blue aidition . 80 Experiment 7. Catalase addition . . . . 8O Experiment 8. Ketabolic poisons . . . . 8l Experiment 9. Penicillin. . . . . . . Intermediate Products in Amino Acid Dissimila- tiODSo O O O O O O O 0 O O O O O O O O O 82 In lizg Studies . . . . . . . . . . . . . . . . lOl DISCUSSION . . . . . . . . . . . . . . . . . . . . . . 114 SUMMARY. . . . . . . . . . . . . . . . . . . . . . . . 124 LITERATURE CITED . . . . . . . . . . . . . . . . . . . 126 iv Table l. 10. ll. 12. LIST CF TABLES Ammonia production from individual amino acids with cheese cloth-strained rumen fluid Ammonia production from individual amino acids with washed suspensions of rumen microorganisms . . . . . . . . . . . . . . . Ammonia production from amino acid mixtures and individual amino acids with washed sus- pensions of rumen microorganisms . . . . . . Ammonia production from amino acid mixtures and individual amino acids with washed sus- pensions of rumen microorganisms . . . . . . Ammonia production resulting from the meta- bolism of optical isomers of six amino acids Ammonia production from six amino acids in eight hour rumen fluid incubations . . . . . Attempts to increase ammonia production from amino acids incubated with washed suspensions of rumen microorganisms. . . . . . . . . . . The effect of two levels of penicillin, 5' I.U. and 50 I.U./ml., on amino acid dissimi- lation by washed suspensions of rumen micro- organisms. . . . . . . . . . . . . . . . . . The post-incubation concentrations (mcM./mlJ and molar percentages of volatile fatty acids from glutaric acid in vitro studies. . Ammonia production from amino acids at three pH's in 24 hr. rumen fluid incubations . . . Indole and skatole production in rumen fluid and washed cell dissimilation studies. . . . The concentrations (mcM./ml.) and molar per- centages of volatile fatty acids from in vitro studies of amino acid dissimilations in rumen fluid . . . . . . . . . . . . . . . 7O 72 75 75 76 77 85 85 97 LIST OF TABLES (Continued) Table Page 15. The concentrations (mcM./ml.) and molar percentages of volatile fatty acids from in vitro studies of amino acid disSimila— 'tions + 0.2% maltose . . . . . . . . . . . . 98 14. The concentrations (mcM./ml. ) and molar percentages of volatile fatty acids fror in vitro studies of amino acid dissimilations in washed cell suspensions . . . . . . . . . 100 15. Indole and skatole production from in vivo dissimilation studies with DL—tryptOphan . . 107 16. The relative effects on jugular plasma amino acid concentrations of L-lysine addition to the rmen. O O O O O O O O O O O O O O O O 0 110 17. The relative effects on jugular plasma amino acid concentrations of L-arginine addition to the rumen . . . . . . . . . . . . . . . . lll 18. The relative effects on jugular plasma amino acid concentrations of DL-tryptophan addi— tion to the rumen. . . . . . . . . . . . . . ll2 vi Figure 1. LIST OF FIGURES in vitro rumen fluid incubation studies with L—lysine, DL-lysine, DL—tryptOphan and. L'3rginine o o o o o o o o o o o o o o 88 Ultraviolet spectra of the tryptOphan fermentation product from washed cell suspensions and an indole standard. . . . 95 Ultraviolet spectra of the tryptOphan fermentation product from rumen fluid and a skatole standard. . . . . . . . . . . . 94 The levels of ammonia nitrogen in rumen liquor after the administration of three amino acids . . . . . . . . . . . . . . . 102 Paper chromatograms of rumen fluid from in vivo studies with L-arginine . . . . . 105 Paper chromatograms of rumen fluid from in vivo studies with L-lysine . . . . . . 106 vii INTRODUCTION At present, little is known of the ruminal inter- mediary metabolism of amino acids resulting from protein catabolism. A few reports dealt with volatile fatty acid, ammonia and gas production from the in giggg and in 1319 incubation of individual amino acids, combinations of amino acids and protein preparations. However, informa- . tion as to those intermediate products formed in amino acid dissimilations and their possible physiological ef- fect upon the host is scanty. Efforts directed towards managing amino acid dissimilation rates and patterns withh: the rumen offer many practical nutritional applications. Three of the more important applications are the following. The studies on the nature of acute bloat in ruminants in- dicate that a toxic substance, or substances, is involved in inhibiting the eructation. Pasture usually results in high ruminal ammonia concentrations which in turn affect the utilization of the herbage magnesium. Lysine and methionine supplementation have been found to increase the nutritional value of certain ruminant rations. Discrepancies have appeared in the literature from studies involving in zitgg rumen liquor and washed cell 'suspensions. In addition, there is a complete lack of knowledge regarding the inability of in ziggg amino acid studies to duplicate the reaction rates noted in vivo. -1- 2 As a result of the foregoing considerations, this investigation had a fourfold purpose. First, catabolic reactions of amino acids were compared and quantitated in both in zitgg,mediums--rumen liquor and washed cells. Secondly, each amino acid was examined chromatOgraphically for intermediary products which could have pronounced physiological activity within the host. Third,-certain materials were added to washed cell suspensions in an ef- fort to increase the amino acid dissimilation rate. And lastly, arginine, tryptOphan and lysine were studied in gizg to compare in iiggg and in gigg dissimilation patterns. REVIEW OF LITERATURE Protein Catabolism In the past fifteen years there has been consider- able interest in the nitrogen metabolism of ruminants and the role of rumenpmicroorganisms in protein digestion. The nitrogen metabolism of ruminants is unique in that much of the ingested feed protein is broken down by micro- organisms present in the rumen prior to entering the major area of amino acid absorption--the small intestines (McDonald and Hall, 1957). A portion of the resultant degradation products are used, in turn, by these same organisms to synthesize microbial protein. Hence the original amino acid composition of the ration may be mark- edly converted to the amino acid composition of microbial protein, which frequently is nutritionally superior to the ruminant (Williams and Hair, 1951). This marked inp tervention of the rumen microorganisms in the nitrogen metabolism of the host also permits a partial substitution of nonprotein nitrogen for protein nitrogen in the ration. For instance, urea can effectively replace natural protein in feedstuffs without affecting growth rates and milk yields, provided the protein replacement with urea does not exceed one-third of the total protein of the ration (Briggs, 1947, Owen, 1941). 4 Studies based on nitrogen balances and nonprotein nitrOgen substitutions for protein nitrOgen in rations, led many investigators to believe that total protein rather than protein quality was the prime essential in the protein requirements of ruminants. However, it is now realized that irrespective of protein breakdown and synthe- sis in the rumen, the nature of the ingested material is also of major importance (Ellis 33 al., 1956). Sym (1958) was the first to show active proteolysis by suspensions of both rumen bacteria and protozoa, as well as by extracts of acetone powders of these micro- organisms. The peptidase activity, however, was weak in his preparations and little free protease was detected in the supernatant rumen liquor. Pearson and Smith (19450) demonstrated proteolysis by rumen microorganisms in 13329. After the incubation of casein or gelatin with rumen con- tents, there was a definite evidence of protein degrada- tion whereas blood meal showed no degradation, but rather a net protein synthesis occurred. These results were at- tributed to a difference in solubilities among the pro-‘ teins since blood meal unlike gelatin and casein is largab' water insoluble. This problem was further examined by McDonald (1948, 1952) who showed that large quantities of ammonia are produced from several different protein souraxs in the rumen under normal feeding conditions. Ammonia proved to make up the major portion of the nonprotein nitrogen fraction. Lewis (1957) stated that numerous factors influence the value of protein to the ruminant other than its di- gestibility and amino acid composition. Such factors as protein solubility, degree of denaturation, particle size, the previous ration of the animal, other nitrogenous com- pounds such as urea and amino acids, and the protein level of the ration influence ammonia production in the rumen. McDonald (1954) was one of the first persons to demonstraua quantitatively that a large portion of the feed protein added to the rumen was converted to microbial protein. He found that about 40 per Cent of the zein given to a sheep was converted to microbial protein. McDonald and Hall (1957) have shown that about 90 per cent of the casein, which constituted 87 per cent of the nitrogen intake, was digested in the rumen in a relatively short time.. These calculations were based on figures obtained by determining protein alkali-labile phosphate; however, it is not neces- sarily true that the release of inorganic phosphate from casein occurs at the same rate as its degradation. An in- teresting anomaly is that the biological value of casein, though high for a monogastric animal because of its high digestibility and favorable amino acid composition, is low for a ruminant. Head (1955) compared the nitrogen reten- tion of sheep receiving casein or fish meal as the protein source in a hay and starch ration, and found that raising 6 the nitrogen intake by raising the casein level of the ration increased the nitrogen balance very little in com- parison with an increase after a similar rise in the level of the fish meal supplement. Chalmers and-Synge (1954) also showed that lambs grew better on fish meal than on casein. Both groups of workers attribute the low nitrogen retention of casein to a rapid and excessive rumen deaminae tion and an increased urinary nitrOgen excretion. In addi- tion, it has been shown that if casein is denatured with formaldehyde so that its solubility is reduced, its nutri- tive value for the ruminant is increased, presumably by a curtailment of ammonia production in the rumen (Chalmers 23 al., 1954). McDonald (1952) and Annison g§,al. (1954) both reported that free ammonia was rapidly formed from casein and ground nut meal, but that flaked maize or its component protein, zein, resulted in low rumen ammonia levels. Fontaine 23 a1. (1944) have suggested that the relative ammonia production in the rumen from standard * feed proteins can be estimated by the prOportion of their total nitrogen soluble in a molar sodium chloride solution under a standard set of conditions. In feeding experiments, two main interrelationships have been demonstrated between carbohydrate and protein :materials in the rumen. There is a better utilization of ‘proteins in the presence of added carbohydrate (Lewis and :McDonald, 1958) and as the protein level is increased, there is a more rapid attack upon the fibrous components of the ration (Burroughs and Gerlaugh, 1949; Burroughs 23 21., 1949; Burroughs gt al., 1950). The effects of high protein rations on ruminal volatile fatty acids have been investigated in sheep (El-Shazly, l952a;Annison, 1954) and in lactating dairy cattle (Davis 23 a1., 1957). Increased concentrations of the total volatile fatty acids occurred when high protein rations were fed, in which the relative proportion of acetic acid decreased and of butyric and' higher acids increased. Further evidence was provided by in gitgg studies (El—Shazly, l952b;Annison, 1956) wherein casein was incubated with washed suspensions of rumen microorganisms. The resultant mixture of volatile fatty acids obtained in both instances was considerably higher in C4 and C5 branched-chain acids than was normally found in rumen liquor. Weller 33 a1. (1958) have analyzed the rumen con- tents of sheep slaughtered at different times after feediq; a ration of Wheaten hay and found that between 2 and 24 hours after feeding, the microbial nitrogen accounted for 63 to~82 per cent of the total nitrogen; soluble nitrogen accounted for 5 to 10 per cent; the remainder being plant nitrogen. This study along with those previously mentioned (McDonald, 1954; McDonald and Hall, 1957) provide ample evidence that feed protein is converted to microbial pro- tein in the rumen in marked amounts. The question then 8 arises as to the intermediate steps in this process. Protein is degraded by the action of the proteo— lytic enzymes of the rumen microorganisms; peptides and amino acids are produced which, in turn, are attacked by deaminases to give ammonia. The demonstration of peptides and amino acids in the rumen, as intermediates in protein digestion, is of recent origin. Lewis (1955) demonstrated that free amino acids could be detected to a greater ex- tent in concentrated rumen fluid 5 hours after feeding than before feeding, and Annison (1956) showed that de- tectable quantities of both alpha-amino nitrogen and diffusible peptide-nitrOgen werwa always present, their concentration increasing up to five- or tenfold after feeding. Furthermore, Annison (1956) found about 2 to 5 per cent of the total microbial nitrogen to be due to amino acids either bound on the cellular surface or exist- ing endogenously. These bound amino acids constituted a substantial part of the free amino acids in rumen liquor and were comprised mainly of those amino acids that have been shown to be most readily dissimilated by rumen micro- organisms. Further information on the proteolytic enzymes of ruminal microorganisms was provided by recent in 11239 studies (Annison, 1956; Warner, 1956). Washed suspensions of rumen bacteria, when used in similar concentrations, caused proteolysis at similar rates irrespective of the ration of the animal. This phenomenon differs from deami- nation since El-Shazly (1952b) found that the capacity of rumen bacteria to deaminate amino acids was prOportional to the readily attacked protein in the ration. In addi- tion, the proteolytic enzymes in 31332 appeared to be rela- tively resistant to environmental changes and to be unal- tered by toluene or acetone. The deaminases were inacti- vated by these organic solvents; the peptidases were inter- mediate in their resistance to inactivation by the usual enzyme isolation techniques. About half the proteolytic activity of the microorganisms taken from the rumen of a sheep on a high protein ration was due to the protozoa and about half due to the bacteria. The Optimum pH of the bacterial proteolysis was between 6.5 and 7.0. The most active bacterial preparations were obtained 6 to 10 hours after feeding the donor animal. Considerable quantities of amino acids, peptides and ammonia were always produced from casein, ground nut cake, and soya protein, but not from bovine albumin, zein or wheat gluten. At present, no ruminal proteolytic activity has been attributed to enzymes secreted by the ruminant itself. In addition, very little proteolytic activity can be demonstrated in cell free ru- men liquor (Warner, 1956). Further investigation is needed with regard to the kinetic properties and specificity of the proteolytic en- zymes, the factors governing the susceptibility of proteins 10 to attack, the intermediates of proteolysis and the effect of these intermediates upon proteolysis. Warner (1956) and Blackburn and Hobson (1960a) have measured the proteolytic activity of rumen bacteria and protozoa. The fractions were obtained by differential. centrifugation of rumen contents from sheep. After wash- ing, each fraction was added to incubation flasks contain- ing buffered casein under toluene. The fractions-~whole fluid, protozoa, large bacteria, small bacteria-~were naturally somewhat mixed, but each contained a preponder- ance of the designated organisms. These results showed that proteolytic activity appears to be distributed over the whole range of rumen organisms. Little activity oc- curred in the cell-free supernatant. The demonstration of proteolytic activity of rumen protozoa is difficult since it is almost impossible to get a preparation of protozoa completely free from bacteria. Attempts to free protozoa of bacteria by washing resulted in a rapid death of the protozoa and a loss of enzymatic activity. Excellent in- direct evidence for protozoan proteolysis was obtained by two means. In a number of experiments using stained pro- tein particles some oligotric protozoa Were observed to ingest and digest these particles. Secondly, experiments employing simple freezing and thawing resulted in a con- siderable amount of active protease from a suspension of rumen protozoa contaminated with a few bacteria, but very 11 little protease was obtained from a similar suSpensiOn of rumen bacteria only. The proteolytic enzymes of washed suspensions of rumen bacteria were stimulated by cysteine (Warner, 1956; Blackburn and Hobson, 1960), resembling in this respect the anaerobic clostridia (Weil £3 31., 1959). Other re- ducing agents such as ascorbic acid or sodium sulphide were without effect as was magnesium ions; ferrous ions stimulated proteolysis for one group but was inactive for the other. These proteolytic enzymes appeared to be rela- tively unaffected by oxygen. All these results suggest that a number of differ- ent rumen organisms play a part in proteolysis. The re- sults of attempts to isolate proteolytic bacteria have not, however, confirmed this. A few microsc0pic observations on the possible proteolytic bacteria in rumen contents have been made. Van der Wrath (1948) and later Warner (1956) noted increased concentrations of gram-positive coccobacilli, bacilli and large cocci in association with casein suspended in the rumen. Masson (1950), observing particles of casein in the general mass of rumen contents, found that they were surrounded by streptococci, and Gall §§_gl. (1951) noted masses of gram-positive cocci in the .rumen of a sheep getting casein as the sole protein. jBurroughs ggpgl. (1950) also noted chains or clumps of cocci in the rumens of cattle getting two rations high in 12 casein. Appleby (1955) studied three sheep on two different rations with the express object of isolating proteolytic bacteria and found that in spite of using an anaerobic technique nearly all the organisms were facultative anaer- obes, the isolated organisms belonging to the genera Bacillus, Micrococcus, Clostriduim and Flavobacterium. The most abundant proteolytic species being Bacillus types, with Bacillus licheniformis predominating. The medium used was made solid with agar and was initially cloudy be- cause of the casein present. After growth, colonies of organisms with extracellular proteolytic properties could be recognized by the clear zones they produced in their vicinity. Bacillus spores were present in large numbers on the food of the animal and were shown to germinate in the rumen. It appears as though most of the isolates may have been derived'from the environment or feed of these sheep. Hunt and Moore (1958) isolated a Flavobacterium sp. from the rumen which behaved similar to the proteinases of some aerobic organisms studied by Weil 33 a1. (1959) but which was inhibited by cysteine. The presence of num- bers of facultatively anaerobic proteolytic bacteria may, of course, help to explain the comparative stability of rumen proteinases to air. Byrant and Small (195%3) found Butyrivibrio giggi- solvens, a common rumen inhabitant, to be proteolytic and 13 this type of organism may play an important role in rumen proteolysis. Gutierrez (1955) isolated in pure culture proteolytic gram-positive bacteria, and Byrant and Burkey (1955a, b) isolated proteolytic gram-positive and weakly proteolytic gram-negative bacteria from the rumen. Only one out of eleven strains of Selenomonas ruminatum tested by Byrant (IU956) was proteolytic when tested with casein. Amino Acid Metabolism The literature on amino acid metabolism is volumin- ous and provides ample evidence of the striking advances which have been made since the isolation of the first amino acid one hundred and fifty-five years ago. Aspara- gine and cystine, the first two amino acids to be identi- fied were discovered in 1806 and 1810, respectively (Vanquelin and Robiquet, 1806; Wollaston, 1810).. Although the existence of 80 amino acids has been demonstrated, there are only 22 amino acids which may be said to occur frequently in protein hydrolyzates. Knowledge concerning the intermediary metabolism of the amino acids has deve10ped from a variety of experi- mental procedures and observations. Pure nutritional studies have provided important evidence leading, in the long run, to the identification of metabolic reactions. The use of mutant strains of microorganisms has proved to be an excellent tool in the study of metabolic pathways, 14 either in the biosynthesis or dissimilation of amino acids. Mutants blocked at various stages of biosynthesis and dis- similation accumulate intermediates, which may often be detected by their growth effects on other mutant or non- mutant strains. Human mutations, alcaptonuric and phenyl- pyruvic oligOphrenic patients, have also been studied, and as in the case with microorganisms, have led to a better knowledge of normal metabolic pathways. Considerable in- formation has resulted from studies with perfusion mechan- isms, tissue slices, tissue homogenates and extracts, and purified enzymes. Isotopes have been widely used in stu- dies of intermediary metabolism and this technique has provided both initial clues and final proof of the occur- rence of many biochemical transformations. There is little doubt that species differences oc- cur in the metabolism of amino acids--for example, the absence of biochemical pathways in mammals when compared to microorganisms, or between microorganisms themselves. 0n the other hand, many pathways are essentially the same among widely different species. As a result, microbes may be used to study animal mechanisms and vice versa. Like- wise, the reactions leading to the synthesis and degrada- tion of amino acids are frequently, but not always differ- ent . Since the rumen is an anaerobic or facultatively anerobic environment, the major portion of this discussion 15 will be limited to amino acid pathways demonstrated by rumen preparations and to pathways demonstrated in strict anaerobes or facultative anaerobes. Detailed investiga- tions of amino acid metabolism by rumen microorganisms are of a relatively recent origin. In 1952, E1 Shazly (1952bh working with whole strained rumen fluid and washed cell suspensions, reported that the main reaction products ob- tained from the incubation of rumen bacteria with casein hydrolyzate were ammonia, carbon dioxide and volatile fatty acids. Analysis of the volatile fatty acids, by gas-liquid partition chromatOgraphy, revealed that strannuz and branched chain 02 to CSfatty acids were present. When the concentrations of the fatty acids produced by incuba- tion of rumen liquor with casein hydrolyzate were compared with the concentrations normally present in rumen fluid, an increase in the branched chain C4 and C5 fatty acids was observed. Paper chromatographic analysis of the re- action mixtures indicated a general decrease in the con- centration of all the amino acids, with a complete disap- pearance of phenylalanine, tyrosine and proline. A new spot consistently appeared on the chromatograms and was subsequently identified as delta aminovaleric acid. 0n incubation of rumen liquor with proline alone, there was no obvious disappearance of proline, nor was any delta aminovaleric acid formed. But, upon incubation of proline and alanine with rumen liquor, both decreased markedly in 16 concentration and delta aminovaleric acid was produced. Alanine, incubated alone with rumen liquor, simply showed a decrease in concentration. 0n the basis of these re- sults, El Shazly concluded that delta aminovaleric acid was formed by a Stickland reaction between alanine and pro- line. He further postulated that the amino acids valine, leucine and isoleucine act as hydrogen donors, while pro- line serves as a hydrogen acceptor in the fermentations with casein hydrolysate. Thus, valine, leucine, and iso- ,leucine would be oxidized to the branched chain 04 and C5 volatile fatty acids, and proline reduced to delta amino- valeric acid. He also pointed out that other reducible substances may function in the place of proline if carbo- hydrate is being fermented simultaneously. A comparison of washed cell suspensions versus ru- men liquor indicated very similar relative rates of de- gradation, however, the deaminating power of washed cell suspensions was closely related to the diet of the animal. In the washed cell studies, the rough equivalence of moles of ammonia, carbon dioxide and volatile fatty acids pro- duced to moles of amino acid destroyed was striking. Sirotnak 33 El. (1955) using washed cell suspen~ aions, reported that decarboxylation and deamination oc- curred only with aspartic acid, glutamic acid, serine, arginine, cysteine and cystine. Arginine and cystine were incompletely utilized after 72 hours dissimilation while 17 the other four amino acids were nearly completely utilized. In each case, volatile fatty acids were produced and hydro- gen sulfide gas was produced from cystine and cysteine. A further observation was a significant enhancement of both decarboxylation and deamination in the presence of maltose. In 1955, Lewis (1955) examined further the deamina- tion of individual amino acids by washed cell suspensions. In this case, significant deamination occurred only in the presence of L-aspartic acid and L-cysteine, although a small production of ammonia occurred in the presence of L-alanine, L—glutamic acid, L-serine and L-threonine. In studies using mixtures of amino acids, it was clear from the results obtained that there was no definite group or number of amino acids necessary to bring about rapid deam- ination. Since the rate of attack upon single amino acids in gixg was considerably more rapid than when washed cells were used, Lewis felt that the method of preparation of the suspension in some way rendered inactive the enzymes responsible for the deamination. Thicker suspensions were used, other redox agents were tested, greater care was taken to maintain anaerobiosis and the effect of adding a portion of ammOnia-free Seitz-filtered rumen contents was investigated. Ammonia production from most of the amino acids when tested individually, however, still was compar- atively slow. 18 Labeled casein dissimilation was studied with washei cells by Otagaki 23 91. (1955). Unfortunately, 85% of the labeled carbon appeared in glutamic and aspartic acid, and no appreciable quantities of isotope appeared in the so- called essential amino acids. Fatty acids accounted for 21%; carbon dioxide for 7%, and microbial protein for less than 0.5% of the recovered activity, respectively. Studies with glutamate -l-C14 and leucine -5-Cl4 were also undertaken. Nearly V5 of the total activity of the former was recovered as carbon dioxide; an unexplainably high Cl4 content (12%) was recovered in the volatile fatty acid fraction, since carbon atom one would be expected to be lost preliminary to any alteration of the five carbon skelton. Studies with 01402 indicated the activity in the volatile fatty acid fraction was not due to carbon dioxide fixation. About fifty per cent of the administered leucine isotope was recovered in the volatile acid fraction, ten per cent in the cellular material and almost none in the carbon dioxide fraction, indicating the main end product of leucine degradation is volatile fatty acids. Lacoste g5 $1. (1958) reported the marked conversion of phenyl- alanine to phenylacetic acid by enriched cultures of rumen microorganisms. The identification of the amino acids valine, pro- line, leucine, and isoleucine as the components from auto- lyzed yeast, casein hydrolyzate and alfalfa extract which 19 increased cellulose digestion 1; 11212 (Bentley 3; 21., 1954; MacLeod and Murray, 1956) lead Dehority 33,31. (1957, 1958) to study their metabolism in order to elucidate their function in this process. Valine and proline were found to have an additive effect on cellulose digestion, leucine or isoleucine could replace valine, but not proline, indicat- ing that the action of valine, leucine, and isoleucine were similar. This led to the idea of a two component system, consisting of a branched-chain component from valine, leucine, or isoleucine and a straight-chain com- ponent from proline. A subsequent study by Dehority 21 51. (1958) presented evidence that valine is oxidatively deam- inated and decarboxylated by rumen microorganisms 1p 11332 to isobutyric acid, and that proline undergoes reductive ring cleavage and deamination at the delta position to form valeric acid. In addition, the proposed intermediates in leucine and isoleucine oxidative deamination and decar- boxylation metabolism were found to possess cellulolytic activity for rumen microorganisms 1g E1332, thus support- ing these proposed metabolic pathways. Both indole and skatole have been reported in rumen contents but little has been done about the mechanism of their production. Spisni and Cappa (1954) found the indohe content of the rumen to be variable and stated the indole could either be of plant origin or arise by bacterial ac- tion on the tryptOphan of the ration. Cappa (1956) 20 investigated the indole content of the rumen in order to explain the presence of indican in bovine urine. He found in the rumen liquor of 15 animals indole levels ranging from 0.09 to 5.0 (av. 0.75) mg./1. Conochie (1955) has reported indole and skatole in the milk of ruminants feed- ing on Lepiduim sp. Nearly saturated solutions of indole (0.1%) and skatole (0.025%) are highly toxic to rumen protozoa, particularly the Isotrichia, Dasytrichia and Ophryoxcolex (Eadie and Oxford, 1954). Investigations by Dougherty gt Q1. (1949) revealed the presence of toxic compounds in the rumen liquor of sheep suffering from acute indigestion. Intravenous in- jections of this rumen liquor into an anesthetized dog or sheep induced a pronounced and prolonged drop in blood pressure along with decreased rumen motility in the sheep. A subsequent study by Dain 93 a1. (1955) identified hista- mine and tyramine as the toxic constituents in the rumen ingesta of experimentally over-fed sheep. Fatal histamine levels exceeding 70 mcg. per ml. were obtained. The histam mine content increased as the rumen acidity became lower than pE5 and reached its highest level below pH4.5, normal sheep ingesta was essentially free ofihistamine. Dougherty (1942) has likewise reported histamine in the rumen liquor of three steers which had died of bloat. Rodwell (1955) isolated eight species of Lactobacilli from the rumen of an over-fed sheep which were able to decarboxylate «r; 21 histidine and form histamine. Shinozaki (1957) identified histamine and methylamine in the rumen liquor of most of the sample taken from animals fed ladino clover or pasture grazed. On rare occasions putrescine and ethylamine were identified. Histamine concentrations were from 2.9 to 5.6 mcg. per ml. on an average; histamine was produced in greater amounts on pasture than on hay rations. High con- centrations of histamine infused into a rumen pouch ap— peared not to be absorbed; this was also true for high dosages of histamine administered via the rumen fistula. Washed suspensions of the LC microorganism, a gram- negative coccus isolated from the rumen of a sheep, fer- mented L—serine, L-threonine, and L-cysteine with the for- mation of ammonia, hydrogen, carbon dioxide and volatile fatty acids (Lewis and Elsden, 1955). Acetone powders of this coccus deaminated both serine and threonine but not cysteine indicating that in this organism, cysteine de- sulphurase is distinct from the enzyme which deaminates serine and threonine. Walker (1958) has found in the LC organism that one enzyme appears to attack both serine and threonine. Perhaps, the dissimilation of aspartic acid by ru- men.microorganisms has been more thoroughly studied than for any other one amino acid. Two groups of workers (Sirotnak 32 21., 1954; Van Den Hands 93,31., 1959) have attempted to elucidate completely the degradative route 22 or routes of aspartic acid. Three major points of dis- crepancy appear between the two groups. Sirotnak (1954) excluded fumarate as an intermediate in the dissimilation of aspartate because fumarate was only decarboxylated and no increase in volatile fatty acids occurred. However, under the experimental conditions of Van Den Hande 22 Q1. (1959) fumarate gave the same end products as aspartic acid; thus it was concluded by these authors that fumarate is an intermediate in the fermentation of aspartic acid. Sirotnak's data indicated to him that malate and oxalo- acetate were not intermediates, but that volatile fatty acids arose only from the decarboxylation of succinate to prepionic acid and the interconversion of prOpionate to acetate and butyrate. Whereas Sirotnak found ethyloxalo- acetate not to be fermented, Van Den Hande found that oxaloacetate gave the same end products as malate. Van Den Hande believed the main pathway of aspartic acid metabo- lism to be via fumarate to succinate to propionate, and did not account for acetate and butyrate from propionate but rather by another minor pathway from fumarate to malate to oxalacetate to pyruvate and then to acetate and butyrate. At the present, limited information is available on the dissimilation of amino acids in the rumen pg; g3. Hueter (1958) comparing 1g 11332 and 1Q 1112 techniques, reported deamination of DL aspartic acid, DL lysine and 23 mixture of DL alanine and glycine. Five individual amino acids, beta-alanine, DL aspartic acid, L—glutamic acid, glycine and L—lysine were fed to a steer by Looper 23 a1. (1959). Beta—alanine, DL aspartic acid and L-glutamic acid were all readily deaminated whereas glycine and L-lysine failed to raise ammonia levels over that of the control. At present, amino acids appear to be poorly ab- sorbed from the rumen (Tsuda, l956b;Annison, 1956). In reality, little is known about the deaminating bacteria in the rumen. In fact, only a few of the pro- teolytic organisms isolated from the rumen produce ammonia (Appleby, 1955). Byrant 23 Q1. (1958) have reported that some strains Of Bacteroides ruminicola produced ammonia. The deaminating ability of the "LC" organism has been mentioned previously (Lewis and Elsden, 1955); however, this Organism occurs in greater numbers in young than in Older animals (Hobson 23 g1., 1958). Dohner and Carlson (1954) reported an interesting case of symbiosis between two strains of Escherichia 9911. Pure cultures failed to ferment lysine, but the two together did so, to give am- monia and butyric and acetic acids. Most of the rumen organisms that exhibit deaminating power are gram-negative (Hobson, 1959); Phear and Ruebner (1956) studying intes- tinal organisms from man found that ammonia production by gram-negative bacteria was greater than by gram-positive Ones. A number of rumen bacteria have been shown to grow 24 on one or more amino acids as sole nitrogen sources, but some rumen bacteria need growth factors, which appear to be peptides and which are found in casein hydrolysate, e.g., Bacteroides ruminicola (Byrant g3 a1., 1958). The following is a brief resume of amino acid me- tabolism by strict and facultative anaerobes in pure cul- tures; however, this resume is meant only to define spe- cific biochemical reactions and is not a complete review on the subject. Each amino acid will be discussed indi- vidually. Glycine. Brasch (1909a, b) reported in cultures of Clostridium lentoputrescens a reductive deamination of glyb cine by molecular hydrogen yielding acetic acid and ammonui Cellular suspensions of Q1. tetanomorphum (Wood and Clifton, 1957), Q1. sporogeneg (Stickland, 1955b;WOOds, 1954; HoOgerheide and Kocholaty, 1958), Q1. histolyticum (Bessey and King, 1954), Q1. welchii (Woods, 1942), Q1. tetani, Q1. botulinum A and B (Clifton, 1942), Q1. 9113;: mentans and g1. sordelli (Prevot g3 g1,,1947), Q1. agggg— butylicum, Q1. butylicum, Q1. flabelliferum, g1. saccharo- but licum, Q1. codophilum and Bacillus Egggg (Nisman 33 a1" 1948a)do not deaminate glycine. The same negative results have been reported for several other anaerobes (Nisman and Thouvenot, 1948). Another anaerobe whose washed suspensions have been reported to deaminate glycine is Diplococcus g1ycinOphilu§ 25 (Cardon, 1942). This organism has been studied in detail by Cardon and Barker (1946, 1947) who showed that the only compound attacked of any appreciable nature by this bac- terium was glycine. Serine and pyruvate were poorly at- tacked in the presence of glycine. The balance sheets of the dissimilation Of glycine by washed suspensions Of this bacterium correSponded to the following equation: 4 CH NH - CODE 4- ZH2O—O4NH5 + 5 CH3 - CODE 4- 2C0 2 2 2 These same authors (1947) reported that only dipep- tides in which the carboxyl group of glycine was free were dissimilated. The only compound containing two adjoining peptides which was degraded was hippurylglycine. Under these conditions hydrogen is a product of the degradation Of glycine. The experiments carried out by Barker, Volcani and Cardon(l948) with Cl# revealed that about 75% of the methyl and 54% of the carboxyl group Of acetic acid arOse from the methylene carbon Of glycine. These Observations indicated that one of the principal reactions Of this fer- mentation is the condensation Of two molecules of glycine or one Of its derivatives through the methylene groups. The two terminal carbons Of the resulting compound, which maybe a C,+ dicarboxylic acid, were converted mainly to . carbon dioxide, the two central atoms being oxidized to acetic acid. In addition, at least 6% of the methyl carbon and 58%iof the carboxyl carbon of the acetic acid arose 26 from carbon dioxide. Glycine then was not directly con- verted to acetic acid. The fact that acetic acid was not metabolized eliminated the possibility that the fixation of carbon dioxide observed involved the following series Of reactions: CO 4. OH5 - coon + 002—9 on} - co - CODE -—-3 COOH - 032 — + 6H' CO - COOH—->2 CH3 Such a mechanism would have produced an effective redistri- bution Of labeled carbon in the acetate molecule. A strain Of Achromobacter oxidized glycine aerobi- cally to ammonia and hydrogen peroxide (Paretsky and Werkman, 1950); presumably the pathway was similar to that which occurs in mammalian tissues and involved glyoxylate and formats as intermediates (Campbell, 1955). Stadtman (1958) has presented evidence that a quinone Operates in the overall process of glycine reduction to acetic acid and ammonia by Clostridium §tick1andii; this same quinone does not appear to Operate in proline reduction to delta amino- valeric acid. Alanine. Brasch (1909a, b) reported for alanine re- sults analagous to those Obtained for glycine with 91. lento utrescsns, i.e., alanine was reduced to prOpionic acid and ammonia. Negative results for the dissimilation of alanine by suspensions Of various anaerobic organisms have been reported by numerous investigators (Woods and 27 Clifton, 1957; Stickland, 1955a;Bessey and King, 1954; Woods and Trim, 1942; Clifton, 1942; PrevOt 93 11., 1947; Nisman 23 a1., l948a;Nisman and Thouvenot, 1948). Cardon (1942) isolated the organism, Clostridium prOpionicum which has been studied in detail by Cardon and Barker (1946, 1947). Of the several compounds assayed, only DL-alanine, DL-serine, DLpthreonine, pyruvate, DL- ‘1actate, acrylate and L-cystsine were fermented by this bacterium. The results of the analysis of the products from the degradation of alanine by cultures of this organ- ism in a medium where the Only organic constituents were alanine and yeast autolysate corresponded to the following equation: - CHNH 5GB - COOH + 2H20-—)2CH - CH - COOH + CH - 2 5 2 3 + 5NH 5 COOH + CO2 5. The dissimilation of alanine appears to be mainly a pro- pionate fermentation. Serine. Numerous anaerobic and facultative anaero- bic organisms attacking this amino acid have been descrflxfl. Cultures of 91. lentOputrescens deaminate serine yielding prOpionic and formic acids, ammonia and other products which were not identified (Brasch, 1909a, b). Washed suspensions of Q1. tetanomorphum (Woods and Clifton, 1957), Q1. prOpionicum (Cardon, 1942; Cardon and Barker, 1946, 1947), Q1. botulinum (Clifton, 1959), Q1. tetani (Clifton, 28 1942; Pickett, 1941), Q1. bifermentans and Q1. sordelli (Prevot 23 11., 1947), Q1. perfringens (Woods and Trim, 1942; Chargaff and Sprinson, 1945a, b)deaminated this amino acid. Chargaff and Sprinson (1945a, b), utilizing Q1. psrfringegg, demonstrated the formation of pyruvic acid from serine in the presence of arsenite. Furthermore, they demonstrated that the alcoholic hydroxyl group of serine must be free for the dissimilation to occur, DL-O- ethylserins, L-phosphoserine and phosphatidyl serine were not attacked. These facts suggested to these investiga- tors that the mechanism of this reaction involved an ini- tial dehydration followed by a hydrolysis of the resulting imino acid. A D-serine dehydrass was obtained from Escherichia 9211 which was activated by pyridoxal phos- phate (Mstzlsr and Snell, 1952a, b). 0n the other hand, an L-specific serine deaminase prepared from E. 3211 was not activated by pyridoxal phosphate but was activated by glutathione and adenylic acid (Wood and Gunsalas, 1949). Cardon and Barker (1947), employing g1. prOpionicum, noted the following catabolic scheme for serine: 5CH20H - CHNH2 - COOH 4» H20 —-)'CH - COOH + 2C0 - CH - COOH + 5 2 2GB + 5NH 5 2 5' Clifton (1940hL employing washed suspensions of Q1. botu- linum type A, suggested that the degradation Of serine by this organism corresponded to the following equation: 29 2CH20H - CHNH2 CH20H + CH5 - COOH. Clifton (1942) studying the degradation of serine by Q1. - COOH + 1120—; 211113 + 2002 + 035 - tetani discovered, in addition to acetic and butyric acids, a significant quantity of alcohol. Cohen 22 a1. (1948) reported the formation of equimolar quantities of acetic and butyric acid from serine with washed suspensions of Q1. gaccharobutyricum, corresponding to the results of Clifton with 91. tetani. The interconversion of glycine and serine is of considerable significance for microorgan- isms and is believed to be folic acid dependent (Lascellss and Woods, 1950; Lascslles 23 a1., 1954; Holland and Meinke, 1949);' Threonine. Woods and Trim (1942), employing sus- pensions Of 91. perfringens, reported a deamination of threonine and the formation of hydrogen, carbon dioxide and ammonia. 'Chargraff and Sprinson (1945aL employing the same organism, isolated alpha-ketobutyrio acid which' resulted from the oxidative deamination of threonine. Cardon and Barker (1947) suggested the following equation for the prOpionic fermentation of threonine by suspensions of Q1. propionicum: 50H - CHOH - CHNH - COOH + Hao—é CH3 - CH2 - CH - 2 2 - COOH + 200 5 n _ - VOOH + 20H; CH2 2 + 3I\H30 Cohen 23 Q1. (1948) reported the production of 2 parts acetic and 1 part propionic acid by Q1. saccharo- 5O butygicum. Pickett (1941), Q1. tetani, and Nisman and Thouvenot (1948), several species of Q1. gporogenes, re- ported the deamination of threonine but did not analyze the volatile acids produced. Barker and Wiken (1948) studied in detail the mechanism of the formation of butyric acid in the degradation of threonine by suspensions of Q1. prOpionicum. They tried to resolve if butyric acid was formed by the condensation of two molecules of acetic acid or one Of its derivatives, as is the case for much Of the butyric acid resulting from clostridial fermentation. To accomplish this, threonine was incubated with washed sus- pensions Of 21. prOpionicum in the presence of acetate labeled with c14 on both carbons. After chromatographic separation Of the mixture Of acids by the method of Elsdsn (1940), almost all Of the radioactivity was recovered in the acetic acid fraction, thereby, excluding the latter as an intermediate in the formation of butyric acid from threonine. Aspartic g211. Brasch (1909a, b) Observed that cul- tures of Q1. lentOputrescens dissimilated aspartic acid to prOpionic and succinic acids. Cohen 33 Q1. (1948) found washed suspensions of Q1. saccharobutylicum fermented aspartic acid producing acetic and butyric acids in the prOportion of B/A equal to 1/1. Cohen-Bazire and Cohen (1949) having noted that this same bacterium fermented 51 oxaloacetate and yielded the same volatile end products, postulated that the degradation of aspartic acid involved first a deamination, then a decarboxylation of oxaloacetic acid ensued which was followed by the dissimilation of the resultant pyruvic acid. Such a postulation was supported by a series of experiments in which clostridia fermented the intermediate compounds of the metabolic scheme (Cohen and Cohen-Bazire, 1948, 1949a, b). The degradation of aspartate has been reported by Woods and Clifton (1957), employing Q1. tetanamorphum, and by Clifton (1942) and Pickett (1941), employing Q1. tetani. In these reports the volatile end products were not determined. Clostridium welchii decarboxylates the beta-carboxyl group of L-aspartic acid to form L-alanine rather than the alpha-carboxyl group (Meister 21 a1., 1951a, b). Small quantities of alpha-keto acids as well as pyrideal phos- phate activate this enzyme. Alpha-keto acids apparently stimulate the formation Of pyridoxal phosphate by trans- amination of the added alpha-keto acids with pyridoxamine phosphate present in the enzyme preparation. This reaction is not a decarboxylation of oxalacetate, which could re- sult from a transamination between the alpha keto acid and aspartic acid, since decarboxylation in the presence of labeled pyruvic acid results in non-labeled alpha- alanine. Crawford (1958) reported an aspartic decarboxyl- ase in Nocardia globerula which was inactive toward all the 32 other amino acids tested. Another pathway Of aspartic acid metabolism is carried out by the microbial enzyme aspartase which reversibly catalyzes the conversion of aspartate to fumarate and ammonia (Woolf, 1929; Erkama and Virtanen, 1951). Glutamic acid. A detailed paper on the degradation Of glutamic acid by suspensions of Q1. tetanomogphum led to the following prOposed overall reaction: SCOOH - CH2 - CH2 - CHNH2 - COOH + 6H20-—-§6CH3 - COOH + 2033'- CH2 - COOH + C02 + 5BR} + H2 (Woods and Clifton, 1958). Barker (1957), employing a Clostridium which was later identified as 91. cochlearium (Barker, 1959), noted the same end products and the same quantitative relationships. Comparable results were obtained by Clifton (1942) with 21. tetani, B/A - 1/2, and by Cohen 22 Q1. (1948) with 91. saccharobut licum, B/A . 1/2. The latter authors identified alpha-keto glutaric acid as the first product Of the reaction. They also verified that 91. saccharo- butzlicum degraded alpha-keto glutaric acid, forming the same acids in the same prOportions. While the initial deamination of glutamic acid was rather rapid, the subse- quent attack Of alpha-ketoglutarate was relatively slow. Krebs (1948) reported a glutamic decarboxylase in Q1. welchii. 55 Valine. The strict anaerobes studied to date are very poor dissimilators of valine as the sole substrate; the negative results obtained may be found in the litera- ture previously cited for negative results with glycine and alanine. Valine has been shown to be decarboxylated by Proteus inilgaris (Ekladius g2 a1., 1957). Further on, it will be found that valine may be deaminated by the Stickland reaction whereby volatile acids are formed. Leucine 1nd Isoleucine. Schmidt, Peterson, and Fred (1924) reported the formation of 1-1eucic acid, (CH5)2 - CH - CH2 - CHOH - COOH, by a hydrolytic deamina- tion Of leucine in cultures Of Q1. acetobutylicum. Other anaerobic organisms have shown little activity towards either leucine or isoleucine; however, both leucine and isoleucine undergo the Stickland reaction. Leucine and isoleucine have been reported to be decarboxylated by Proteus vulgaris (Ekladius 23 11., 1957). Phenylalanine. The strict anaerobes appear to be poor dissimilators of phenylalanine. Several facultative anaerobes have been reported which deaminate phenylalanine to phenylpyruvic acid (Henricksen, 1950). Tyrosine. Brasch (1909a, b) reported that cultures of Q1. lentoputrescens reduced tyrosine to p-hydroxyphenyl- prOpionic acid. Janke (1950) and Rhein (1921) noted the 54 formation of p-cresol with 91. gpesologeneg, a species whose cultures unfortunately are no longer in collections. Rhein (1921) observed the formation of phenol by Q1. tetani and g1. pseudotetan1ggm. Prevot and Saissac (1947) obtained phenol and p-cresol from tyrosine with Inflabilig teras and g1. corallinum, respectively. Histidine. Woods and Clifton (1957) reported the deamination of histidine by Q1. tetanomorphum. Pickett (1941) showed that suspensions of Q1. tetani deaminated histidine with an Opening of the imidazole ring, the volatile acids formed being butyric and acetic acids in the respective proportions of 0.51 to 2.11 moles per mole of histidine fermented. Cohen 33 Q1. (1948) observed an analogous phenomenon with suspensions of Q1. saccharo- butylicum when they obtained a formation of butyric and acetic acid in the prOportion of B/A = 1/2. They proposed that the Opening of the imidazole was due to a histidase analagous to that discovered by Edlbacher and Kraus (1950) in tissues and implied an intermediary formation of glu- tamic acid. Histidine is fermented by Micrococcus aggg- ggngg to ammonia, carbon dioxide, acetate, butyrate, lac- tate, and traces of formats, with urocanate as an inter- mediate product (Whitehmn 1957). Glutamate was also an intermediate in this case. 55 Proline and hydroxyproline by themselves are quite refractive to degradation by suspensions of anaerobiclbac— teria but are attacked in the presence of hydrogen donating amino acids, e.g., alanine, valine, leucine or isoleucine (Stickland, 19553). Tryptophan itself is not deaminated by the strict anaerobes studied to date. HOpkins and Cole (1905) demon- strated that indole and skatole were formed from trypto- phan by E. 9911. The mechanism of the over-all reaction of indole formation was established by Woods 23 Q1. (1947) and was confirmed by Davis and Happold (1949). Two alternate pathways for the complete oxidation of tryptOphan have been reported for microorganisms (Happold, 1950). Both Of these pathways apparently are initiated by the peroxidase- Oxidase system and formylase which leads to the formation of kynurenine from tryptOphan. The presence of kynurenin- ase leads to the aromatic pathway and the formation of anthranilic acid and catechol whereas the quinoline path- way depends on the presence of a transaminase which induces the formation of kynurenic and xanthurenic acid. Cystine and cysteine. Several microorganisms have been reported which cause a nonoxidative desulfhydration with either a simultaneous or subsequent deamination of cysteine (Fromageot, 1951). Woods and Clifton (1957) re- ported the deamination Of cystine and cysteine by suspen- 56 sions of Q1. tetanomorphum. Methionine. Woods and Clifton (1957) and Pickett (1941) have reported the deamination of methionine by Q1. tetanomorphum and g1. tetani. Nisman and Thouvenot (1948), employing g1. sporOgenes, isolated alpha-keto-gamma methiobutyric acid from methionine in the presence of sodium arsenite; mercaptan formation was also noted. The anaerobic degradation in certain strains of Pseudomonas produced ammonia, alpha-ketobutyric acid and methyl mer- captan (Kallio and Larson, 1955). Lysine was decarboxylated to cadaverine by Bacillug cadaveris and E. coli (Gale, 1940) whereas none of the anaerobes studied were found to deaminate lysine (Cohen, 1949). Arginine ang ornithine. These amino acids have been deaminated by a number of anaerobic species; Woods (1956) reported 91. sporogenes reduced ornithine to delta- aminovaleqxic acid (47%) and that arginine also gave rise to delta-aminovaleric acid. Stadtman (1954),working with an amino acid-fermenting Clostridium, observed that 6 per 14 was reduced to delta-amino- cent of ornithine - 2 - C valeric acid and 1 per cent was converted to proline. Ar- ginine and ornithine are decarboxylated to agmatine and putrescine, respectively, by E. 9311 (Gale, 1940) and a strain of Lactobacillus (Rodwell, 1955). 57 The Stickland Reaction Stickland (1955a) reasoning from the earlier experi- ences of Knight and Fildes (Stickland, 1955» on the nutri- tion of Q1. sporogenes, discovered that the energy neces- sary for the growth of this bacterium resulted from a re- action between amino acids. Stickland placed amino acids and Q1. sporOgenes in Thunberg tubes with methylene blue or brilliant crssol blue as oxidation-reduction indicators. He found that alanine, valine, leucine and pyruvate were good donators of hydrogen. Utilizing the leucoderivative of benzyl violet, Stickland Observed that the following amino acids were hydrogen acceptors: glycine, proline and hydroxyproline. Then he placed both a hydrogen donating amino acid and a hydrogen accepting amino acid along with washed bacterial suspensions into Thunberg tubes and mea- sured the deamination. By this means, Stickland demonstra- ted that two amino acids were deaminated whereas each one individually was not. The results of Stickland (1955) for alanine and glycine may be represented by the follow- ing equation: 2CH2NH2 - COOH + CH5 - CHNH2 - COOH + 2H20 --4) 50H COOH + 5NH + CO . 5 5 2 The importance of such a reaction to strict anaerobes, which do not utilize oxygen as a hydrogen acceptor, is readily apparent. When proline served as the hydrogen acceptor, Stickland (l955a)observed that the reduction Of 58 proline was made without deamination, giving rise to delta aminovaleric acid. Neuberg (1911) and Ackerman (1907, 1908,1909) had previously discovered delta amino- valeric acid in mixed cultures of putrefactive bacteria. Stickland (1955 aDprOposed that the oxidation of ala- nine occurred as follows: (1). CH - CHNH - COOH + 2H20—I’ CH COOH + C0 + NH 4- H2 5 2 5 2 5 Initially alanine would be converted to pyruvic acid: (2) CH3 - CHNH2 - COOH 4- H2O—’ CH5 - CO - UOOH + NH3 + H2 Pyruvic dehydrogenase would then produce acetic acid as follows: (5) CH -CO-COOH+HO-—DCH -COOH+CO 4-H 5 2 5 2 2 Reaction (1) then represents the sum of reactions (2) and (3). _ The reduction of glycine utilizes the 2H2 formed during the oxidation of alanine and is brought about by deamination: (4) 2NH2CH2 - COOH + 2112—)2CH5 - COOH + NH5 The Stickland reaction then is represented by the sum of reactions (1) and (4). In the presence of 10"5 molar sodium arsenite the Stickland reaction is totally in- hibited, however, Nisman and Vinet (1949h)have demonstrated that in the presence of this inhibitor and methylene blue as the hydrogen acceptor, the oxidation Of the hydrogen donating substrate is carried out normally, whereas the oxidation by glycine of the leuOOphenosafranine is inhibitaL 59 Therefore, two distinct enzymes appear to be Operative in the coupled deamination. Woods (1954) demonstrated that ornithine and argi- nine are also hydrOgen acceptors. Their reduction gave rise to delta aminovaleric acid. Valine and leucine were shown by Stickland (1955a1to be good hydrogen donators; Hoogerheide and Kocholaty (1958) added isoleucine. These authors, however, did not conduct experiments comparable to that which Stickland (1955d}made for alanine. With this in mind, Cohen-Bazire g5 g1. (1948a, b) performed ex- periments designed to better elucidate the dissimilative patterns of valine, leucine and isoleucine in the Stickland reaction.‘ Prevot and Zimmes (1946) had established that 91. valerianicum and Q1. caproicum produced respectively an acetic-valeric and acetic-caproic fermentation when they Were grown on meat broth and 10% glycogen.) Cohen- Bazire g1 g1. (1948a, b), however, Observed the same fer- mentations in a non-glucose medium and surmised that va- leric and caproic acids were not catabolites arising from the degradation of glucose. Only acetic acid was formed when glucose or pyruvate were substrates. These authors then concluded that isobutyric, isovaleric and valeric acids were arising from the branched chain amino acids valine, leucine and isoleucine. Washed suspensions of Q1. caproicum and g1. valerianicum did not deaminate these amino acids when they were used as individual substrates, 40 but deamination did occur in the presence of hydrogen ac- cepting amino acids, either glycine or proline. The bal- ance sheet of Cohen—Bazirs (1948a, b) permitted the follow- ing equation to be written for valine: (0115);, - CH - CHNH - COOH + 21:11 on - COOH + H20——> 2 2 2 (CH3)2 - CH - COOH + 2CH5 - COOH + 5NH5 + C02 The exactness of the prOposed catabolic equations was veri- fied by the ammonia levels, the measured volatile acidity, the analysis of the nature and prOportion Of the acids formed and the man om etric measure Of liberated carbon dioxide. Proline proved to be a better hydrogen donator than glycine for these analyses patterns of valine, leu- cine and isoleucine in the Stickland reaction. Prevot and Zimmes (1946) had established that 91. valerianicum and Q1. caproicum produced respectively an acetic-valeric and acetic-caproic fermentation when they were grown on meat broth and 10% glycogen. Cohen-Bazire 33 Q1. (1948a, b), however, observed the same fermentations in a non-glucose medium and surmised that valeric and caproic acids were not catabolites arising from the degradation of glucose. Only acetic acid was formed when glucose or pyruvate were sub- strates. These authors then concluded that isobutyric, isovaleric and valeric acids were arising from the branched chain amino acids valine, leucine and isoleucine. Washed suspensions of Q1. caproicum and g1. valerianicum did not deaminate these amino acids when they were used as 41 individual substrates, but deamination did occur in the presence Of hydrogen accepting amino acids, either glycine or proline. The balance sheet Of Cohen-Bazire (1948a, b) permitted the following equation to be written for valine: (CH3)2 - CH - CHNH2 (CH3)2 - CH - COOH + 2GB - COOH + 2NH - COOH + H O--9 2032 - COOH + 5NH 2 + CO 5 5 2 Proline proved to be a better hydrogen donator than gly- cine for these analyses due tO the formation of DOD! ‘volatile delta aminovaleric acid. Optically active valeric acid was not definitely established as the volatile acid arising from isoleucine due to inherent errors in the ana- lytical technique employed, but the acid Obtained proved not to be Optically active caproic acid which could have arisen by a reductive deamination Of isoleucine. Earlier, Neuberg and Karczag (1909) had described reductive deamination by mixed cultures of bacteria wherebyr isovaleric acid arose from valine and Optically active ca- proic acid from isoleucine. Optically active caproic acid has been reported in tobacco leaf sap (Sabety and Panouss, 1947) and in petroleum (Quebedaux g; 11., 1945). TO date, however, there has been no evidence in the literature Of reductive deamination of valine, leucine and isoleucine by anaerobic bacterial suspensions (Cohen-Bazire g; 31., 1948a, b). Wagner 21 Q1. (1925) Observed a marked accumu- lation of ammonia and volatile acids in cultures of Q1. botulinum grown on peptons broth. The volatile acids were 42 a mixture of acetic, butyric and valeric acids. Clifton (l940a)reported that 91. botulinum was capable of perform- ing the Stickland reaction and thought that the valeric acid fraction Of Wegner g2 Q1. (1925) could have arisen from the deamination of delta aminovaleric acid by certain strains of 91. botulinum. At present, it is known that the valeric acid fractions reported by earlier workers was probably a mixture of Optically active valeric acid, iso- valeric and isobutyric acids and these acids originated from branched amino acids via the Stickland reaction. In addition to Q1. gporogeneg, Q1. botulinum, Q1. valerianicum and g1. caproicum, Nisman g5 g1. (1948a, b) have extended the Stickland reaction to the following spe- cies: g1. histolyticum, g1. flabelliferum, Q1. saprotoxi- cum, g1. sordelli, Q1. bifermentans, 91. butyricum, Q1. acetobutylicum and Inflabilis indolicus. Q1. saccharobu- t licum, g1. tetani, g1. iodophilum, Q1. tetanomorphum, Q1. pgyfringens and 1. Egggg were incapable of performing the Stickland regardless of the pair of amino acids stu- died; this was also true for the following facultative anaerobes: Staphylococcus aureus, Proteug vul aris, Klebsiella pneumonia and Escherichia coli. It would ap- pear then that the enzymes of the Stickland reaction are limited in general to the proteolytic species of the Clostridiacea. Species giving the Stickland reaction pro- duce a mixture of acetic, C4 and C5 acids whereas other 45 species have an acetic-butyric fermentation. The latter acids therefore arise from the degradation of glucose and the deamination of those amino acids which give principally acetic and butyric acids; this group constitutes the major- ity of the Clostridiaceae. Raynaud and Macheboeuf (1946) reported that the Stickland reaction carried out by Q1. sporOgenes is inhib- ited in the presence of glucose. Cohen-Bazire g3 g1. (1948a, b) and Saissac 21 Q1. (1948) reported this same phenomenon for other species. Nisman and Thouvenot (1948) demonstrated that 91. aerofoetidum, Q1. carnofoetidum, g1. mitelmani and g1. goni possessed the enzymes of the Stick- land reaction. Nisman 33 Q1. (1948b) reported that in this reaction glucose, pyruvate, acetaldehyds and ethanol, but not lactate and succinate, could replace the hydrogen do- nating amino acids; glycine, for example, was just as well deaminated in the presence of these compounds as in the Presence Of alanine (Prevot and Zimmes, 1946). It has been demonstrated recently that the strict anaerobes possess enzymatic systems capable of utilizing oxygen as a hydrogen acceptor (Rosenberg and Nisman, 1949). Umier aerobic conditions, Nisman and Vinet (1949) observed tfluit amino acids of the hydrogen donor group were converted maiJaly to the corresponding fatty acids; however, small quantities of the corresponding alpha-keto acids were fOr‘msd. These authors found that, under aerobic conditions, 44 the amino acids of the hydrogen acceptor group and oxygen competed for the hydrogen liberated from the amino acid donor» There was evidence that diphosphOpyridine nucleo- tide was involved in the carrier system (Nisman, 1954; Mmmelok and Quastsl, 1955); it was demonstrated that di- phosphopyridine nucleotide might be reduced by alanine and that the reduced coenzyme could undergo reoxidation by proline or glycine. Non-Protein Nitrogen The knowledge Of the ability of rumen bacteria to utilize nonpprotein nitrogen for their own protein needs dates back to the nineteenth century. For it was in 1891 that Zuntz (1891) prOposed the utilization of amides and ammonium salts as precursors Of ruminal bacterial protein and the subsequent digestion.and.assimilation of this prO- tein.by the host. However, it was the impetus Of the scarcity of nitrogenous concentrates in Germany prior to $41 during World War I and the development of processes for the synthesis Of non-protein nitrogen compounds that led German scientists to be very active in research pro- Jects dealing with the usage of urea and'other non-protein nitrogen materials as protein substitutes in ruminant ra- 1310us. From these studies and the many subsequent inves- t1Sations which followed, certain conclusions have been I‘eached regarding those conditions suitable for the most 45 efficient utilization of urea nitrogen. Pearson and Smith (19450)and Mills g; g1. (1944) have shown that the degree Of protein synthesis is related to the amount and type of carbohydrate present in the ration, starch being the most efficient. 1gby1§gg studies by Wegner 23 11. (1940) dem- onstrated the importance of the level of protein in the ration on the utilization of urea nitrogen. These workers found that urea utilization was lessened when the total protein concentration of the rumen ingesta exceeds 12 per, cent. Later work by Johnson 31 Q1. (1942) and Hamilton gt Q1. (1948) showed that the nutrient value of rations for growing lambs was diminished when less than 16 per cent Of the total nitrogen is in the form of preformed pro- tein. Loosli and Harris (1945) reported in lambs both higher absolute amounts of nitrogen and higher percentages 0f absorbed nitrogen on 10 per cent protein rations con- taining urea plus methionine than on urea alone. In 1944, Johnson 23 Q1. (1944) defaunated the rumen of sheep by feeding copper sulfate and showed that it was the bacteria and not the protozoa in the rumen which were reSponsibls for protein synthesis from urea. Defaunating the rumen with cOpper sulfate did not reduce the ability or sheep to utilize urea. Smith and Baker (1944) like- wiSe demonstrated that protein synthesis from urea was nOt I‘educed in the absence of the rumen protozoa. These au- thOrs further stated that the chief contributors to protein 46 synthesis are the small rods, cocci and vibrios of the microiodophilic pOpulation. Mann g3 Q1. (1954) and Mackay and Oxford (1954) succeeded in isolating gram-positive micrococci, and a gram-negative rod from the rumen which were urease-positive and facultatively anaerobic. In ad- dition, Gibbons and Doetsch (1959) have isolated from the rumen a gramépositive, nonmotile, asporogenous, pleomorphic rod resembling Lactobacillus bifidus which hydrolyzed urea and was obligately anaerobic. The urease in rumen fluid resembles in activity that from soya and jack beans in that changes occur in its activity with changes in temperature and pH and its behavior in the presence of inhibitors as quinone and sodium fluoride is typical of enzymes of the urease type (Pearson and Smith, 1945). These authors found the inhibition Of urease by quinone could be prevented by thiol compounds such as hydrogen sulfide and cysteine. Ruminants appear to differ from nonruminants in bl00d urea metabolism, a phenomenOn which is largely attri- butable to the rumen microflora. In the ruminant, urea ffirmed by the liver moves via the blood or saliva into the rumen,where it is utilized for protein synthesis. The sub- sequent digestion and absorption of protein in the small intestines constitutes a mechanism whereby protein nitrogen C811 be continually regenerated. Of course, the regeneramon 47 rate would be governed by the urinary urea excretion rate. Here again the ruminant shows a uniqueness since Schmidt- Nielsen 23 Q1. (1957, 1958) has demonstrated that the mechanism of urea excretion in ruminants differs from that Observed in other mammals. This conclusion is based on their findings that camels and sheep, when placed on a low protein ration, decreased the fraction of filtered urea appearing in the urine to very low values. It was found in the camel that low protein intake caused the urea clearance to decrease to values of only 1 to 2% while the urea fil- tered in the glomeruli rose to 40% when a normal ration was fed. The authors also observed in both the camel and sheep that the kidneys continue to conserve urea for some time after nitrogen intake is increased following a pro- longed period of low nitrogen intake. This regulation was found.to be independent of the glomerular filtration rate, Plasma urea-concentration and osmotic load and therefore the regulation appears to be on the tubular level (Schmidt- Nielson 21 31. 1958). These authors further stated that theirdata was consistent with the previOusly suggested 'mfpothesis "that the excretion of urea in the mammalian k1daisy is brought about through a regulated active trans- POI‘t of urea, accentuated by a counter-current multiplier System represented by Henle's loop and vasa recta." In- deed, the urinary urea excretion rate appeared to be gov- erned by the nitrogen intake and growth rate. 48 When known quantities of urea were injected intra- venously into protein depleted sheep fed a low nitrogen, high carbohydrate ration, only about 52% of the injected urea could be recovered in the urine (Houpt, 1959). How- ever, the utilization Of injected urea decreased to a mean of 22 per cent when dietary carbohydrate was withheld dur- ing the urea injection studies. In experiments in which the isolated rumen of anesthetized sheep was emptied and replaced with warm physiological saline, Houpt found the total urea -N transfer to the rumen to be 5.2 mmoles urea -N/hr. This value was from 40 to 67% lower than the rates found in the urea injection experiments (7.8 - 15.0 mmole/ hr.) and was attributed by the author to the experimental conditions. The 7.8 to 15 millimoles/hr. reported utilized by Houpt in the rumen agrees well with the 8.6 mmol. urea -N/hr./4 liter rumen reported by Pearson and Smith (1945cL In the rumen saline experiment on sheep, 5.5 to 16 times as much urea passed directly from the blood to the rumen as moved with saliva. McDonald (1948) has estimated that the salivary urea nitrogen in the sheep contributes at least 0.5 g. nitrOgen to the rumen daily which is somewhat higher than the value Of 0.28-0.56 g. given by McDougall (1948). Somers (1958) also found 0.29 g. urea nitrOgen in the daily salivary secretion of sheep. Unfortunately, accurate data is not available on the urea nitrogen contributed to the bovine rumen, but calculations based on urea concentrations 49 Of 10-15 mg. % and an average salivary secretion of 85-90 liters per day would be 8.5 to 11.7 g. per day (Phillipson and Mangan, 1959). Another interesting facet of urea metabolism in ru- minants was studied by Tsuda (l956a, b) employing a Pavlov pouch in a goat's rumen. In attempting to elucidate whethmr urea is absorbed directly from the rumen, Tsuda placed three concentrations of urea solutions 0.2, 1 and 5 per cent into this miniature rumen. Only at the 5 per cent level did appreciable absorption take place; and since when urea is fed to ruminants, the concentration in the rumen fluid is generally below 0.5 per cent, it would appear that direct absorption of urea through the rumen wall does not occur under practical feeding conditions. In summary, urea transfer appears to be a one way mechanism from the peripheral blood system or saliva to the rumen which occurs in both normal and stressing cOndi- tions. The unique urea conservation mechanism Operative in the renal tubules of the ruminant serves to continually re- generate nitrogen so essential tO the rumen microbial flora during periods Of low nitrogen intake. Ammonia is a key intermediate of microbial nitrogen metabolism in the rumen. Rumen ammonia is derived from several sources: (1) a breakdown Of feed protein, (2) a deamination of free amino acids in feedstuffs, (5) a pro- teolysis of the rumen microorganism, (4) a hydrolysis Of 5O feed, salivary and ruminal urea, (5) a breakdown of ammo- niated feedstuffs and (6) a reduction of feed nitrates. In contrast to the relatively high level Of ammonia in the ruminal and portal veins of the ruminant, the con- centration of ammonia in the peripheral blood, other body fluids, and the tissues of ruminants is very low (McDonald, 1948). McDonald (1948) calculated that ammonia absorption from the sheep's rumen could amount to about 4 to 5 g. am- monia nitrogen per day when ruminal ammonia levels aver- aged 25 mg. per cent. Normally, the ammonia concentration of rumen contents approximates 8 to 40 mg. per cent and reaches a maximum at about 2 to 5 hours after feeding (Head, 1959). Lewis, 23 Q1. (1957) demonstrated a close correla- tion between changes in rumen-ammonia levels and the levels Of ammonia in the portal blood. NO regulatory mechanism for the adsorption of ammonia appeared to exist; ammonia transfer seemed to be one Of simple diffusion. Excessive levels Of ammonia may appear in the peri- pheral circulation whenever the urea synthesizing capacity Of the liver is exceeded and in such a case, is deleterious to the animal. Toxicity studies have revealed that the rumen ammonia concentration at which ammonia appeared in the peripheral blood was not constant. Head and Rock (1955) Observed peripheral blood ammonia at rumen ammonia concen- trations as low as 50 to 40 m. moles per liter, while Annison 25,11. (1957) reported rumen ammonia concentrations 51' as high as 85 m. moles per liter without noticing any toxic effects. A level of l to 2 mg. per cent of ammonia in the peripheral blood of ruminants produces toxic symptoms (Repp 22 21-. 1955; Lewis 21 s1.. 1957; Dinning ai.sl.. 1949). Pearson and Smith 15mm) and Smith 23; 11. (.1956) ob- . served a marked stratification in the ammonia concentra- tion of rumen ingesta. In both instances, a higher con- centration of ammonia occurred in samples taken from the tOp ingesta than in samples from the bottom. Reis and Reid (1958) reported that the Optimum pH for ammonia pro- duction from casein in the rumen varied between 6.0 and 637 on different rations; ammonia production fell rapidly on the acid side on the Optimum pH and less rapidly on the alkaline side. 2 Ammonia is a key intermediate in the dynamic state Of mammalian protein metabolism. Schoenheimer and his as- sociates found that when N15 (as ammonia or amino acids) was given to rats, the isotOpe subsequently appeared in almost all of the amino acids (Foster 22 11., 1959; Rittenberg 33 11., 1959; Schoenheimer g§,g1., 1959a, b). In general, examination of the body revealed that the amino acid originally fed exhibited the highest concentra— tion of isotope followed in order of isotOpe concentration by glutamic and aspartic acids. When N15 aspartic acid was fed, the isolated glutamic acid had the greatest concentra- 52 tion of isotOpe. _ Two amino acids, lysine and threonine, appear to occupy a special metabolic position in mammals in that they do not appreciably incorporate administered N15 from am— monia or other amino acids. When lysine labeled with deuterium and N15 was fed to rats, N15 was found in other amino acids, but the lysine incorporated into the tissues had a deuterium to N15 ratio which was almost the same as that Of the administered amino acid (Weissmann and Schoen- heimer, 1941; Ratner e; 1.1., 1945). Similar findings were also noted with threonine (Elliott and Reuberger, 1950). Lewis (1951a) has demonstrated the conversion Of nitrate to ammonia in the rumen and postulated that ni- trite and hydroxylamine were intermediates in this reduc- tion. An accumulation of nitrite in the rumen may occur under conditions whereby large amounts of nitrates are present in the ration. Nitrite then may be absorbed with toxic effects due to a methemoglobinemia. Lewis (1951b) has also shown, using washed suspensions of rumen bacteria, that hydrogen was a very active donor for the reduction of nitrate, nitrite and hydroxylamine. Formats, succinate, lactate, citrate, glucose, malate and mannitol were also hydrOgen donors for nitrate reduction, but less active than hydrogen itself. 55 Protein Anabolism Since the rumen is a dynamic system, the factors affecting the quantity Of protein synthesized in the rumen are numerous and complex. Most of these factors were dis- cussed previously and included the level of protein in the ration, the nature of the ration protein, other constitu- ents in the ration, etc. Knowledge about the extent Of this protein synthesis and processes involved are essen- tial for the understanding of the contribution of this phenomenon to the nutrition of the ruminant. This section then will include the magnitude of protein synthesis within the rumen and a few specific examples of the anabolic pro- cesses. It has recently been reported (Gray 33 31., 1958) that in sheep fed on Wheaten hay the amount of nitrogen reaching the abomasum and passing on to the duodenum was equivalent tO almost 100 per cent of the nitrogen ingested; in other words there was no overall loss of nitrogen from the rumen by absorption. Since subsequent studies (Weller 32 31., 1958), employing diaminOpimelic acid for the assay, revealed that throughout the entire day 61 to 82 per cent of the nitrOgen in the rumen was present as microbial ni- trogen, it would appear that this range represented the extent Of the conversion Of plant nitrogen to microbial nitrogen. 54 Pearson and Smith (1245's) and McNaught and Smith (1947) estimated the extent of microbial protein produc— tiOn in the mature cow to be 100 to 500 grams per day. Duncan 33 31. (1952) found 19 to 190 per cent more protein in the rumen six hours after feeding a purified ration con- taining urea as the sole source of nitrogen. Agrawala 33 31. (1955) demonstrated a 55 to 109 g. increase in true protein in the rumens of six month Old calves fed a puri- fied ration and a 252 g. increase on a natural ration. Lysine synthesis by rumen bacteria has been demon— strated by McNaught (1951) and Edwards and Darroch (1956). McNaught demonstrated a 12 per cent increase in the lysine content of 13,31333 fermentation flasks whereas Edwards and Darroch demonstrated the ruminal synthesis of lysine by feeding a ration devoid of lysine to lactating goats and demonstrating lysine containing proteins in the milk. These latter authors also calculated that the ruminal synthesis of protein in these goats amounted to 40 g. per day. Allison 33 31. (1959) incubating Rum1ngcoccus £1333- faciens in the presence of isovalerate - 1 - 014 recovered radioactive leucine from protein hydrolyzates of this or- ganism. Block 33 31. (1951) and Emery 33 31. (1957a) de- monstrated the incorporation of radioactive inorganic sul- fate into cystine and methionine by rumen bacteria. The latter authors found that the fOrmation of cystine was about twice as great during the three-hour incubation 55 period as the formation of methionine. When examining the response of single strains of bacteria, Emery 33 31.(195flfl noted that only five out of ten strains studied utilized significant amounts of inorganic sulfate in the synthesis Of organic sulfur compounds. EXPERIMENTAL PROCEDURE ' 13 Vitro Studies The donbr animals for these studies were mature cows, weighing from 1050 to 1200 pounds, fitted with the screw cap, plastic fistula plugs described by Hentschl 33 31. (1954).. These animals were fed rations of 6 to 10 pounds Of alfalfa hay and 10 to 14 pounds of a concentrate mix- ture, composed Of 77.5% ground shelled corn, 20% soybean Oil meal, 1% calcium carbonate, 1%idica1cium phosphate and 0.5% trace mineralized salt. The concentrate mixture also contained 5000 international units of vitamin A and 450 international units Of vitamin D per pound. The crude pro- tein content was 15.8%. All the cows received 50 grams Of trace mineralized salt daily. The experimental rations were fed once daily at 7 a.m. for at least three weeks be- fore the investigations were initiated. The animals had free access to water. General methods. Samples of rumen fluid which were to serve as inocula were collected at 5 to 4 hours after feeding. The rumen ingesta was strained through a double layer of cheese cloth to remove all extraneous solid ma- terial. In those studies which are designated as rumen liquor incubations the material described above served as the incubating medium. The amino acids were dissolved in 56 57 200 m1. Of rumen liquor in amounts equivalent to 0.01M solutions. The washed suspensions of rumen bacteria were prepared in the following manner. The cheese cloth strained material was centrifuged in an International cen- trifuge Size 2 model V for 5 minutes at 250 x G to remove the large feed particles and protozoa. The resulting supernatant fluid was then subjected to a force of 27,600 x G for 10 minutes in a Serval centrifuge type SS-l. The supernatant was discarded and the bacterial residue was suspended in 25 m1. of 0.1M phosphate buffer at pH 6.5, previously boiled and cooled, to which was added 0.02% (w/v) Na2S . 9H20. This material was again centrifuged at 27,600 x G for 10 minutes; the supernatant was discarded and the bacterial residue resuspended in the above buffer. Three parts of the bacterial suspension (dry weight, 4 to 7 mg./ml) were then added to ten parts of the buffer con- taining the amino acid or amino acids. Quantities of amino acids equivalent to 0.01M solutions in 15, 150, and 260 ml. total volumes were dissolved in the buffer solu- tions just prior to the addition Of the bacterial suspen- sion. Each amino acid was incubated alone and in combina- tion with two and three other amino acids in rumen liquor and washed cell preparations. Whenever tryptOphan was studied, the amino acid was solubilized in 5 m1. of 1N Na OH prior to the additiOn of the rumen liquor or phosphate 58 buffer. Whenever aspartic or glutamic acid was studied, the amino acid was neutralized to pH 6.9 with sodium car- bonate prior to incorporation in the incubation medium. Anaerobic conditions were Obtained by gassing for five minutes with carbon dioxide freed from oxygen by bub- bling the gas through a chromous acid solution. The rumen liquor incubations were conducted for 8 or 24 hours at 59° 0., whereas the washed cell suspensions were incubated at 59° C. for 8, 24 or 48 hours. Samples for amino acid chromatOgraphic analysis were obtained by adding 0.5 ml. of the amino acid medium to 2 m1. of absolute alcohol just prior to incubation (zero hour) and“at the termination of the experiment. Samples for ammonia and volatile and nonvolatile acids were taken just prior to incubation (zero hour) and at the terminatrmi of the experiment. These samples were preserved by adding 1 ml. of 50% (v/v) sulfuric acid to 50 m1. of sample. This volume of acid lowered the pH to 1.5 to 2.0 and yielded a final sample in which most Of the suspended solids were precipitated. Samples arising from incubation studies with tryptOphan were preserved by adding 1 cc. of a satur- ated solution of mercuric chloride to 59 cc. Of the incu- bating medium. These samples were stored at 6° C. until analyzed which was always less than a month. The Keeney column (1955) was used for the determi- nation of volatile fatty acids. Ammonia was determined by 59 aeration or steam distillation from an alkaline sample. The ammonia was collected in a 2% solution of boric acid and titrated to the mixed Kjeldahl indicator (100 mg. methyl red + 50 mg. methylene blue) endpoint. Glutaric acid analysis. Paper chromatography-was used as a semiquantitative measure of glutaric acid dissi- milation. A series of glutaric levels of 5, 10, 15, 20 and 25 mcM./ml. were added to rumen fluid and washed cell endogenous preparations to serve as standards. Twenty-five micromoles of glutaric acid per milliliter previously neu- tralized with 1N NaOH was used in the duplicated 24-hour rumen fluid and washed cell dissimilation studies. At the end of 24 hours, 15 ml. were taken from each of the dissi- milation and standard solution flasks and steam distilled under acid conditions to remove the volatile acids. The 'samples were then evaporated on a hot plate to a volume of 5 ml. The 5 ml. was extracted successively with three 10 ml. portions of peroxide-free ethyl ether. The ethyl ether was evaporated on a hot plate and concentrated to a final volume of 5 ml. One hundred microliters Of the ethyl ether extract was chromatographed onto Whatman no. 1 filter pa- per. Descending chromatography was employed using ethyl alcohol, water and ammonium hydroxide (80:10:10). The pa- per was air dried and sprayed with a 0.04% solution of bromophenol blue in 95% ethyl alcohol adjusted to a defi- nite blue color (pH 6.7 as determined with a glass elec- 60 trode) with dilute sodium hydroxide (Buch 33 _1., 1952). Another spray reagent employed with a mixture of 2 g. glu- cose, 2 cc. aniline, 20 cc. water, 20 cc. ethanol, and 60 cc. butanol (Bastie, 1957). The sheets were placed in an oven at 115° C. for 10 minutes. The acid spots appeared dark brown on a pale yellow background. Concentration dif- ferences of 5 ch./ml. were readily detected. 33133 3313 analysis. The amino acids and their dissimilation products were identified by unidimensional descending paper chromatography. A number of the well- known amino acid develOping solvents were used for this purpose: (1) butanol, acetic acid, water (4:1:1); (2) pyridine, acetic acid, water (50:55:15); (5) phenol, water (80:20); (4) ethyl alcohol, butanol, pyridine, water (60: 10:5:25); (5) isobutyric acid, water (80:20). Leucine and isoleucine were separated with tertiary amyl alcohol, pro- panol, and water (4:1:1). Cadaverine and putrescine were separated with phenol, water (80:20) in a 0.5% ammonia at- mosphere with 100 mg. of sodium cyanide in 4-6 ml. of water at the bottom of the chamber. » Thirty microliters of the sample was spotted on Whatman no. 1 paper. The chromatograms were air dried over night. The separated amino acids were visible as fluorescent spots under ultraviolet light. The chromato- grams were sprayed with 1% ninhydrin in 95% ethanol and developed in the dark at room temperature. 61 Since Rf values may undergo fluctuations, the un- knOwn ninhydrin-reactive compounds were identified by com- paring the suspected known, the unknown and a mixture of the suspected known and unknown compounds on the same chromatogram. This technique compensates for any devia- tion of the Rf value in the unknown caused by extraneous material. Repeatable results, by this technique, in three to four different solvents constituted identification of the unknown. Analysis 31 tryptophap derivatives. Indole and skatole were identified by means of paper chromatOgraphy using isOpr0pyl alcohol, 28% ammonia, water (10:1:1). The chromatOgrams were air dried and sprayed with Ehrlich's reagent. This reagent is of particular value in that it reveals both the indole structure and compounds containing free amino groups. In addition, the colors obtained with different compounds containing the indole structure vary widely and characteristically, as do the time required for these colors to appear. Indole and skatole were likewise identified using the ultraviolet regions of the spectrum Of a Beckman DK-2 ratio recording spectrOphotometer. Indole was isolated by extracting the tryptOphan incubation .medium with three times its volume of peroxide-free ethyl ether. The incubation medium was extracted three times successively with its own volume of ethyl ether. The ethyl 62 ether was dried over anhydrous sodium sulfate and evapor- ated Off in a rotary vacuum flash evaporator into 2 ml. of distilled water. Concentrations were chosen so as to ob- tain a maximum absorbancy for the sample of 0.50-0.85 unit; the absorbancy being controlled by dilutions or reduction ,of the cell path from 1.0 cm. to 0.1 cm. Indole and ska- tole were determined quantitatively using a modification of the p-dimethylaminobenzaldehyde method of Meyers (1950). In this case, the chloroform was evaporated off at 57° C. in a current of air rather than siphoned off. Amine analysis. Samples for amine analysis were divided into two portions. The first portion was made alkaline by adding 4% by volume of 10% sodium hydroxide. This sample was then extracted three times successively with its own volume of peroxide-free ethyl ether. -The ethyl ether was evaporated off in a rotary vacuum evapora- tor into one ml. of 2NEhEu Such a procedure should have resulted in a 50 to 100 fold concentration, depending upon the Original volume of the sample selected. The second portion was chromatographed directly. 13 Vivo Studies The animals used in these studies were the same ma- ture fistulated cows used in the 13 vitro studies and were receiving 12 lb. of alfalfa hay and 8 1b. of the previously described concentrate mixture. The experimental ration 65 was fed once daily at 7 a.m. for at least three weeks be- fore the investigations were initiated. The animals had free access to water. In the 13 3133 amino acid dissimilation studies the endogenous, Lplysine and DL-tryptOphan studies were per- formed using the same animal whereas the L-arginine study was performed on another animal, The two animals, however, were of similar body size and were fed identical rations. For purposes of calculating the amount of each amino acid to be administered via the fistula, it was assumed that each animal weighed 1200 1b., the rumen contents consti- tuted 14% of the total body weight and the rumen contents were comprised of 85% water, by weight. Using these fig- ures, the rumen would contain 65 liters of aqueous phase. Equivalent amounts of DL—tryptophan, L-arginine . H01, or L-lysine . HCl were dissolved in a liter of water so that the 65 liters of aqueous phase in the rumen contained the equivalent of a 0.02M solution of amino acid. The level of amino acid amounted to 265.5, 274, and 257 grams of DL- tryptophan, L—arginine . HCl and L—lysine . H01, respec- tively. The DL-tryptOphan was solubilized with 800 cc. of 1N NaOH and then made up to one liter with water prior to fistular administration. Each amino acid solution was administered via the fistula two hours after feeding. The amino acid solution was thoroughly mixed with the rumen ingesta by stirring 64 with the arm and fist for five minutes. A portion of the rumen ingesta was then strained through a double layer of cheese cloth to remove the larger feed particles. This rumen liquor served as the zero hour sample. Rumen liquor samples were taken for paper chromatography and ammonia analyses at 0, l, 2, 5, 4, 6, 8, 10, 15 or 14, 25 or 24 hours after the administration of each amino acid. Samples for amino acid chromatographic analysis were obtained by adding 0.5 m1. of rumen fluid to two m1. of absolute alco- hol. Rumen fluid samples in the L-lysine and L-arginine studies were preserved by adding One ml. of 50% (V/V) sul- furic acid to 50 ml. of sample whereas in the tryptOphan studies the rumen samples were preserved by adding one m1. of a saturated solution of mercuric chloride to 59 m1. of rumen liquor. Ammonia was determined by the permutite method de- scribed by Hawk 33 31. (1954) with modifications. Three m1. of rumen fluid were added to a 100 ml. volumetric flask containing two grams of amberlite IR-l20. The mixture was allowed to stand several minutes before the rumen fluid was decanted from the flask and the resin washed with dis- tilled water. Two m1. of a 10% sodium hydroxide solution were added and the flask was allowed to stand again. Afte the addition of 75 ml. of distilled water to the mixture, two drOps Of Gum Ghatti were added followed by 10 ml. of Nessler's reagent. Five minutes was allowed for color 65 develoPment before diluting to 100 ml. The optical density of the sample was then determined at 4’30 millimicrons in a Beckman B Spectrophotometer.‘ A standard curve was prepared in'a similar manner using aqueous ammonium Sulfate solutions. Indole and skatole were determined quantitatively using a modification of the p-dimethylaminobenzaldehyde method of Meyers (1950). Blood samples were obtained by jugular venepuncture O, l, 2 andnq hours after the fistular administration of the amino acid solution. For plasma samples, approximately 40 to 45 ml. of blood were drawn into tubes in which the ' anticoagulant was one ml. of a 10% potassium oxalate solu- tion evaporated to dryness. Fifty ml. of ethyl alcohol were added to five m1. of plasma. After standing for 15 minutes the tubes were centrifuged for 5 minutes at 250 x G to remove the plasma protein. The supernatant was decanted and evaporated to dryness at below 50° C. under vacuum. ‘ The ninhydrin reactive components of the bovine plasma were purified and separated into three fractions by employing the technique of Thompson 23 3;. (1959). The water-soluble residue was taken up in five ml. of water and transferred to a seven cm. column (30 x 1 cm.) of Dowex 50-X4 in the ammonium form. Two 3 ml. washings were like- wise transferred to the above resin. The column of Dowex 50-X4 in ammonium form retained-the basic amino acids and 66 amines. The eluate was allowed to drip onto a seven cm. column (50 x 1 cm.) of Dowex 50-X4 in the hydrogen form which retained the neutral and acidic amino acids. The basic amino acids--arginine, lysine, and histi- dine--were eluted with 80 ml. of 2N ammonium hydroxide. The eluate was dried down under carbon dioxide free con- ditions at moderate temperatures (( 50° C.). The column was washed free of excess KH40H with 40 ml. of deionized water, the ammonium ion was removed from the resin with 50 ml. of 0.50 I 0.02% dCl and the eluate was discarded. The strongly basic amines were eluted with 50 ml. ofEfi.Ilfll “The column of acid resin was treated with small portions of 2N NH40H until the effluent was just basic (8-10 ml.) and then washed with 40 ml. of deionized water. The effluent, containing the-neutral-and acidic amino acids, was dried in vacuum at below 50° C. The three fractions were taken up individually in 5 ml. of 50% alcohol-50% water. One and a half milliliters of this mixture was used in the paper chromatography stu- dies. flhatman No. 5 mm. paper (16%" x 22%") was used for one or two dimensional descending chromatography. The basic amino acids and amines were chromatOgraphed unidi- mensionally employing pyridine/acetic acid/water (50/55/l5L phenol/water (80/20) in a 1% ammonia atmosphere, or ethanob’ diethylamine (77/1). The chromatograms were air dried and 67 sprayed with a 1% solution of ninhydrin in 95% ethanol. The neutral and acidic amino acids were separated employing two dimensional descending chromatography. Phenol/water (80/20) in a 1% ammonia atmosphere was used as the first solvent and n-butanol/acetic acid/water (62/12/26) as the second. The second system contained 0.1% ninhydrin (w/v). The chromatograms were air dried and allowed to develOp overnight in the dark. RESULTS In Vitro Studies The first phase of this project was concerned with the relative rates of dissimilation of amino acids added individually to the two in gitgg,mediumsa-cheese cloth- strained rumen fluid and washed suspensions of rumen micro- organisms. The extent of deamination of each amino acid in cheese cloth—strained rumen liquor are presented in Table l and in washed suspensions of rumen microorganisms in Table 2. The amino acids are arranged in the tables in decreasing order of deamination and can be roughly grouped into three separate classes based on their activity. L- or DL—serine, L—cysteine, L-aspartic acid, L-threonine, and L-arginine were attacked most completely, followed by L—glutamic acid, L-phenylalanine, L-lysine, L-cystine and DL—lysine forming an intermediate group, and a third group in which deamination was much less pronounced was DL- tryptOphan, delta amino valeric acid, L-methionine, L- alanine, L—valine, L—isoleucine, L-leucine, L-ornithine, L-histidine, glycine, L-proline and L-hydroxyproline. Am- monia production from arginine revealed only a 57 to 80% dissimilation whereas paper chromatography on the same samples revealed arginine to be completely dissimilated within 24 hours. Apparent quantities of ornithine, however, 68 69 Table l. Ammonia production from individual amino acids with cheese cloth-strained rumen fluid. (24 hr. incubation; 0.01M soln. of amino acid., The am- monia values represent the total ammonia minus the control value) Ammonia N (mg/100 ml) Theor. Yield Actua % dis- Sample of Amino N , Yield similation2 L-Serine 14.01 ' 15.98 100 DL—Serine 14.01 14.06 100 L-Cysteine 14.01 15.42 96 L—Aspartic Acid 14.01 15.52 95 L-Threonine 14.01 11.66 85 L-Arginine H01 56.04 44.91 80 L-Phenylalanine 14.01 10.52 75 L-Glutamic Acid 114.01 8.90 64 L-Lysine HCl 27.99 16.02 57 L—Cystine 28.02 15.04 47 DL-Lysine 801 27.99 11.54 41 DLpTryptOphan 14.01 5.14 57 L—Histidine 301 14.01 4.50 52 L-Ornithine 28.02 8.04. 29 L—Valine 14.01 4.01 29 L-Alanine 14.01 5.89 28 L-Leucine 14.01 5.42 24 L-Isoleucine 14.01 5.10 22 Delta AVA3 14.01 2.90 21 Glycine 14.01 1.55 10 L~Hydroxyproline 14.01 1.26 9 L-Proline 14.01 1.18 8 I Mean of three values. 2Range 3 10 per cent. 5Delta amino valeric acid. 70 Table 2. Ammonia production from individual amino acids with washed suspensions of rumen microorganisms. (24 hr. incubation, 0.01M soln. of amino acid. The ammonia values represent the total ammonia minus the control values) Ammonia N (mg/100 ml) Theor. Yield Actual % dis- Sample of Amino N Yieldl similation2 L-Serine 14.01 15.80 99 DL-Serine 14.01 15.90 99 L-Aspartic Acid 14.01 12.10 86 L-Cysteine 14.01 11.86 85 L-Threonine 14.01 9.62 69 L-Glutamic Acid 14.01 7.92 57 L-Arginine H01 56.04 51.82 57 L-Lysine HCl 27.99 10.40 57 L-Cystine 28.02 9.62 54 Delta AVA5 14.01 3.02 22 L-Phenylalanine 14.01 2.98 21 L-Methionine 14.01 2.50 18 D-TryptOphan 14.01 2.52 17 DL—TryptOphan 14.01 2.06 15 L—Histidine HCl 14.01 2.05 14 L-Ornithine H01 28.02 5.92 14 L-Alanine 14.01 1.98 14 L-Valine 14.01 1.54 11 L-Leucine 14.01 1.48 11 L-Isoleucine 14.01 1.59 10 Glycine 14.01 0.52 2 L-Proline 14.01 0.24 2 L—Hydroxyproline 14.01 0.16 1 1 Mean of three values. 2Range 1 10 per cent. 5Delta amino valeric acid. 71 still persisted. Serine, L—cysteine and L-aspartic acid were markedly dissimilated, 80-100%, in 24 hours regard- less of the medium used. The other dissimilatable amino acids were present in variable quantities at the end of 24 hour incubations. The amino acids catabolized in rumen fluid were likewise dissimilated by washed cell suspen- sions and in approximately the same sequence of magnitUde. The dissimilation rates, however, are more rapid and c0m— plete in the rumen fluid studies than in the washed cell suspensions. The one exception was delta amino valeric acid which was not dissimilated at a distinguishable faster rate in rumen fluid. When three or four amino acids were incubated to- gether with washed suspensions of rumen microorganisms, the only phenomenon which differed from amino acids incu- bated alone was the marked increase in the dissimilation of proline and alanine incubated together. The actual yields of ammonia from a mixture of three or four amino acids can be compared to the sum of ammonia production from each amino acid in Tables 5 and 4. In this trial the amount of each amino acid added was the same, 10 mcK./m1., resulting in a final amino acid concentration of 50 to 40 mcM./m1. These results demonstrated that the most readily dissimilated amino acids were responsible for the major portion of the ammonia arising from mixtures of three or four amino acids. Ammonia production and visible 72 Table 5. Ammonia production from amino acid mixtures and individual amino acids with washed suspensions of rumen microorganisms. (24 hr. incubation, 50 mcH./m1., 10 mcH./ml. of each amino acid. Ammonia values represent the total ammonia minus the control value.) Ammonia N (mg/100 ml) Theor. Yield Actual % Dissi- Individual Sample of Amino N Yield milation Yield L—Lysine HCl 15.50 L-Aspartic Acid 56.01 42.18 75 ' 11.70 DL—Serine 15.85 40.85 L—Leucine 1.20 L-Methionine 42.05 4.88 12 2.20 L-Phenylalanine ' 2.55 6.35 Glycine‘1- 0.16 L—Aspartic Acid 42.05 12.90 51 15.10 L-Proline 0.00 15.26 L-Arginine 20.80 DL—Tryptophan 84.06 22.84 27 5.40 L-Valine 1.10 25.50 ,Glycine 0.00 L-Alanine 42.05 5.70 9 2.60 L-Isoleucine 1.56 4.16 75 Table 4. Ammonia production from amino acid mixtures and individual amino acids with washed suspensions "of rumen microorganisms. (24 hour incubation, 40 mck./m1., 10 mu M/ml. of each amino acid. Ammonia values represent the total ammonia minus the control value.) l :— Ammonia N (mg/100 ml) Theor. Yield Actual % Dissi- Individual Sample of Amino N Yield milation Yield L-Aspartic acid . 12.40 L-Threonine 56.04 24.90 44 8.70 L—lsoleucine 1.72 L-Norvaline 1.48 24.50 L-Glutamic 7.60 L-Proline 56.04 16.88 ‘ 50 0.40 L-Phenylalamine 5.24 L-Alanine '2.10 15.34 L-Histidine . 1.84 DL-Tryptophan 112.08 ' 52.68 29 2.42 L-Lysine HCl 10.40 L-Arginine HC1 20. 0 55.56 L-Leucine 1.70 L-Methionine 56.048 5.10 9 2.10 L—Histidine 1.84 L-Proline.«- 0.16 5.80 74 disappearance of the amino acids as noted by paper chroma— tography were closely correlated. A third phase of this study dealt with the metabolism of the Optical isomers of six amino acids. The amino acids employed were serine, tryptOphan, aspartic acid, lysine, threonine and phenylalanine. The results of this experiment are found in Table 5. Ammonia production from both the L form and either the D or DL form was determined. The ammonia levels corresponded well with the amount of amino acid dissimilated as noted by paper chromatography. These results indicate that both isomers of certain amino acids, such as serine and tryptophan, are dissimilated equally as well whereas only the L isomers of certain amino acids, such as aspartic acid, lysine, threonine and phenyl- alanine are readily dissimilated and the D enantiomorph are either catabolized slowly or not at all. Since the peak of ammonia production usually occurs in the rumen at one to three hours after feeding, six amino acids were dissimilated in 11359 using rumen liquor as the incubating medium in order to eluCidate the rela- tive magnitude of ammonia production in 1113 from indivi- dual amino acids. An eight hour incubation period was employed for two reasons. The first was the fact that since only one amino acid served as an ammonia source, an incubation period of greater than one to three hours would be necessary to resolve apparent differences. The second 75 Table 5. Ammonia production resulting from the metabolism of Optical isomers of six amino acids. (24 hour incubation, 10 ch./ml. of amino acid). Ammonia N (mg/100 ml) Amino Acid Isomer Medium Theor. Actual % Dis- Yield Yield similation Serine L rumen liquor 14.01 15.98 100 DL rumen liquor 14.01 14.06 100 Serine L washed cells 14.01 15.80 99 DL washed cells 14.01 15.90 99 Tryptophan DL washed cells 14.01 2.06 15 D washed cells 14.01 2.52 17 Aspartic Acid L rumen liquor 14.01 15.52 95 DL rumen liquor 14.01 8.40 55 Lysine L rumen iquor 27.99 16.02 57 DL rumen liquor 27.99 11.54 41 Threonine L rumen liquor 14.01 11.66 85 DL rumen liquor 14.01 5.82 42 Phenylalanine L rumen liquor 14.01 10.52 75 DL rumen liquor 14.01 6.19 44 was that the incubation period must be short enough to prevent substrate depletion from entering into the results. The results of this eXperiment are found in Table 6. In eight hours arginine contributed approximately five times as much total ammonial nitrogen as did any one of the other five amino acids studied. 76 Table 6. Ammonia production from six amino acids in eight hour rumen fluid incubations (lO mck./m1 of amino acid). Ammonia N (mg/100 ml) Amino Acid Theor. Yield Actual Yield % Dissimilation L-Arginine 56.04 d 24.14 45 L-Aspartic Acid 14.01 5.16 57 L-Serine 14.01 4.52 52 L-Cysteine 14.01 4.59 51 L—Lysine 14.01 2.56 .18 ”DL-Tryptophan 14.01 1.82 15 The final phase of this study dealt with various attempts to increase the magnitude of deamination in washed suspensions of rumen microorganisms. Modifications of the usual washed cell suspensions are given below and the results of each are summarized in Table 7. Experiment 1. Egg 3; Enriched Cultures. Eight in- dividual amino acids, 50 mch./m1., were incubated for 48 hours. The rumen microorganisms were centrifuged out and resuspended in a medium containing the same,amino acid (10 Imch./m1.) with which they had been previously incubated. Experiment 1 reveals that such enriched cultures are very poor dissimilators of amino acids as compared to Experiment 0 in which conventional washed cell suspensions were em- ployed. A slide was prepared from each of the 72 hr. 77 .0H00 ofinmam> oswfim 0pH00 H 0:.m mm.m 0m.¢ mu.m ma.¢ 0:.m 00.0 00.: mqflnpfiQHOIq ms.m 40.0 08.0 0H.m 80.0 00.0 00.0 oa.m eeaqeaeaseegmuq 0H.0 00.u 00.5 08.5 0m.m 00.0 mm.0 00.5 0Ho< 0Hawpsa0lq 80.0 08.4 me.m 0m.m 00.0 om.m 00.0 0o.m emanateswenqm 80.0 $0.0H 05.0H mm.0H 00.HH 00.NH NH.H 0H.ma 0004 oflpnmmm< 0paon se.0 04.4 00.0 40.0 00.0 00.8 m0.o 00.0 eeamsqua 0H.0m $0.0m mm.mm N0.dm $0.0m da.mm m0.H m0.mm onwaamm Hompooo mnp How cmpommuoo comp 0>0n ..Hs 00H\z HmquOasm .08 a4 s0>flm .mpadmon map Ham ”.0 can #0 mqowmeSocfi .93 :m “0800 oqasm mo .Hs\.aos 0H0 msmfiqmmnoopeas smash co 0G0H0n00050 00amms spas 00p0p50mH 00800 coast scam soaposvonm mwaoaam mmmmnosfl 0p mumsmppw .5 manwa 78 incubation flasks and compared with a slide made of the original microbial suspensions. All the slides were gram . stained. The preincubation slide revealed a variety of morphological forms of gram-positive and gram-negative microorganisms whereas the 72 hr. incubation slides re- vealed only gram-negative individual cocci. Experiment g. Eygidoxamine. In order to reduce the ammonia content of endogenous washed cell suspensions, it is necessary to wash the microorganisms several times. This procedure, however, invariably results in a consider- able reduction of activity. Such a phenomenon indicated a loss of cofactors essential in the deamination of amino acids. Experiment 2 was designed to study the effect of added pyridoxamine on the magnitude of deamination of eight amino acids. Pyridoxamine was added in a quantity that re- sulted in a final concentration of 2.5 x 10"5 M. The re- sults of experiment 2 failed to reveal any consistent ef- fect of added pyridoxamine on amino acid dissimilation by washed suspensions of rumen microorganisms. Experiment-5. Eyridoxal Phosphate. Experiment 5 was similar to experiment 2 except that the coenzyme pyri- doxal phosphate was substituted for its vitamin ana10gue pyridoxamine. The pyridoxal phosphate was added in quan— tities equivalent to a final concentration of 2.5 x 10-6 M. The results of experiment 5 also were of a negative 79 nature, failing to reveal any significant effect of added pyridoxal phosphate on amino acid dissimilation by washed suspensions of rumen microorganisms. Experiment 4. .Magnesium igp. Since pyridoxamine or pyridoxal phosphate apparently were not the sole essential cofactor lost, experiment 4 was performed to ascertain if an exogenous source of divalent cations would increase the magnitude of ammonia production from amino acids. Magne- sium ion in the form of magnesium sulfate was added to the incubation medium, giving a final concentration of magneshml ions of 5 x 10'"4 M. The results of the addition of magne- sium ions to washed suspensions of rumen microorganisms were negative. Experiment 5. Potassium igg. In experiment 5 an all potassium buffer was compared with the control buffer (approximately equal amounts of potassium and sodium ions) due to the fact that cellular and extracellular potassium levels influence amino acid uptake by Ehrlich ascites tu- mor cells (Riggs g: a;., 1958). The all potassium buffer had a potassium ion concentration of 0.097 M whereas the control buffer had a potassium ion concentration of 0.066.M and a sodium ion concentration of 0.065 M. In order to maintain these cationic ratios in the control buffer, as- partic and glutamic acids were neutralized with equivalent amounts of sodium and potassium bicarbonate whereas potas- 80 sium carbonate alone was employed in the experimental flasks. This procedure increased the cationic strength but did not alter the molar ratios of sodium and potassium. The ammonia production in each of the two buffer systems was essentially the same, indicating that such an altera- tion in the extracellular potassium level had no apparent influence on amino acid dissimilations by washed cell sus- pensions of rumen microorganisms. Experiment 5. Methylene pig; addition. Washed cell suspensions of rumen microorganisms are highly reductive systems and the possibility existed that amino acid dis- similation rates are slow due to a failure to reoxidize the reduced forms of coenzymes I and II and flavinadeninedinu- cleotide. Methylene blue chloride was added to each incu- bation flask to give a final concentration of 0.1%»methy- lene blue. The results were not consistent but a fifty per cent reduction in ammonia production did result from the addition of methylene blue to L-lysine, L-ornithine, DL- tryptOphan and delta amino valeric acid incubation flasks. Experiment 2. Catalase addition. One ml. of tech- nical grade catalase solution1 was added to the amino acid incubation in an attempt to offset the possible formation of toxic hydrogen peroxide in the dissimilation studies. lNutritional Biochemicals Corporation, Cleveland 28, Ohio. 81 This catalase had a potency of 50 units per ml. which de- composed 75 times its weight of H202. The ammonia produc- tion of the catalase-added flasks and the control flasks were essentially the same, demonstrating that catalase addition to washed cell suspensions of rumen microorganisms was of no benefit in the catabolism of amino acids. Ammonia production and amino acid disappearance as noted by paper chromatography were highly correlated in experiments 1 through 7. Experiment 8. Metabolic Poisons. In an analagous study, an attempt was made to increase the dissimilation rate of serine and thereby produce catabolic end products that would be present in greater concentrations. An eight hour incubation period was employed with L-serine, 10 mcx/ m1. Two metabolic poisons, sodium fluoride and sodium arsenate which block key steps in the glycolytic cycle (Fruton and Simmons, 1959), were used as 0.017% and 0.029 M solutions, respectively. The ammonia levels were 4.52, 5.97 and 6.58 mg. of ammonical nitrogen per 100 ml. for the endogenous, sodium arsenate and sodium f1uoride.incu- bation mediums, respectively. Such results would indicate that arsenate and fluoride in the above concentrations in- hibited the endogenous metabolism of rumen microorganisms. One ml. of 0.2% dinitrophenylhydrazine in 2Niifl.had also been added to each flask in an attempt to isolate the pyruvic acid produced in serine dissimilation. The 82 hydrazone was not found in any of the three cases. It would appear that pyruvate is a very labile substrate in rumen microbial metabolism and as such is very difficult to isolate in the hydrazone form. Another excellent pos- sibility would be that the dinitr0pheny1hydrazine was ex-~ tracellular only whereas the pyruvic acid existed only intracellularly. Experiment 9. Penicillin. The final phase of this study dealt with the effect of penicillin on amino acid dissimilation by washed cell preparations. Three Interna- tional Units (I.U.) of penicillin per m1. had no effect on amino acid dissimilations, whereas a penicillin concentra- tion of 50 I.U. per m1. markedly inhibited amino acid dis- similations, Table 8. Intermediate Products in Amino Acid Dissimilations The second major asPect of this study deals with the intermediary products formed in amino acid dissimilations by rumen microorganisms. This was accomplished by chroma- tographic and spectrophotometric examination of the amino acid fermentation mediums prior to and after incubation. Paper chromatographic analyses of fermentation mix- tures, to which L-arginine, L-ornithine and L-lysine had been added, alone and in various combinations, revealed that these three amino acids decreased in concentration as the fermentation proceeded. All three of these amino acids 85 Table 8. The effect of two levels of penicillin, 5 I.U. and 50 I.U./m1., on amino acid dissimilation by washed suspensions of rumen microorganisms (10 mch./ml. of amino acid; 24 hr. incubations at 59° 0.; all the results, given in mg. ammonical N/100 m1., have been corrected for the control value). ' Substrate Control 5 I.U. 50 I.U. None 0.88 1.04 0.18 L-Arginine 25.56 27.64 0.46 Delta AVAl 5.10 2.56 0.00 L-Aspartic Acid 10.82 10.16 0.16 DL-Tryptophan 5.12 5.18 0.00 L-Glutamic Acid 7.42 8.06 0.12 L-Phenylalanine 5.66 2.26 0.00 5.72 lDelta amino valeric acid. gave rise to further ninhydrin reactive products when in- cubated in either rumen fluid or washed cell suspensions. One of these spots proved to be delta amino valeric acid. In the identification of delta amino valeric acid, it was noted that the new spot corresponded in Rf value to those obtained with a standard of this amino acid. Addition of known delta amino valeric acid to the unknown mixture did not result in the appearance of any new spots when tested This was true using both one- in several solvent systems. and two-dimensional paper chromatography. 84 The formation of delta amino valeric acid from L- lysine, L-ornithine and L-arginine was'much easier to demonstrate in rumen fluid than in washed cell incubations. The appearance of this amino acid d2 pggq was demonstrated in rumen fluid in all ten experiments employing lysine, .ornithine and arginine whereas its presence in washed cell. suspensions was demonstrated only in those incubations which indicated a good dissimilation of the original amino acid and after the incubation mediums were concentrated twenty fold. An attempt was made to ascertain the metabolic pro- ducts formed in the dissimilation of delta amino valeric acid. However, delta amino valeric acid dissimilation was slow in both rumen fluid and washed cell suspensions. Al- though Rothstein and Miller (1955) have shown that delta amino valeric acid is converted largely to glutaric acid in the intact rat, paper chromatographic analyses of post incubation samples of arginine, lysine, ornithine and delta amino valeric acid failed to reveal the presence of glu- taric acid. An experiment was performed to determine if glutaric acid would be dissimilated by rumen microorganisms. In- cubations of 25 mck./ml. of glutaric acid were carried out in rumen fluid and washed cell suspensions. No increase in volatile fatty acids was detected in this one trial, Table 9.. A series of glutaric acid standards of 5, 10, 15, 85 .0060 64409540 .Aopmhpmnsm 00000 .0400 00400540 00 .Ha\.zom mm 0344 04:40 amasm 0o .4a\.aoa 00 0:40 44cc eczema .064p04pc06moo 0Homum>4mm A: phospHBV msoqomo0sm 4406 0mnmmzm . AmDMHmeSm 60.060 PSOQPHBV mdoflmwodflm UflSHH 4.400444%” 00.00 00.00 00.48 00.60 00.04 00.0 40.0 00.0 00.0 66.4 80.6 000.8.6+.6.e 84.00 40.80 ,00.68 60.80 00.04 00.0 80.8 00.4 06.4 40.4 00.6 0.000 .6.4 00.04 00.00 08.0 40.00 44.0 00.04 04.0 60.0 00.6 06.0 00.6 00.8.0+.6.e 80.04 40.00 00.0 00.80 66.0 60.44 88.4 00.8 00.6 60.4 00.6 840.8.0+.6.8 64.04 06.00 08.0 00.00 68.0 00.64 00.4 60.4 00.6 80.0 00.6010e0 4406 000004 00.084 04.00 60.00 80.04 00.00 00.64 00.04 60.0 04.0 68.6 00.6 00.8.0+.0.0 00.084 00.00 06.00 00.04 00.00 00.64 00.04 00.0 00.8 00.6 40.6 040.8.0+.0.0 40.004 04.00 40.864 06.04 06.00 00.64 44.0w 00.0 64.0 00.6 .40.6 4.080 .0.0 me4o8 as o4poo< as o4do4d as o4aae ms o4am4 as no 040880 4aeoe loam -20 nas -0040 .00403pm 6444> mm 0400 Ho mmmwpnmoumm 40468 0mm A.Ha\.2600 64406540 8640 00060 hppmw 044p046> mmo4pmhp206Qoo wo4pwndon4npmom one .0 04084 86 20, and 2S mcm./ml. were processed and spotted chromato- graphically. A comparison of the post-incubation mediums failed to reveal a dissimilation of glutaric acid by rumen microorganisms. In addition to delta amino valeric acid, three other ninhydrinJreactive products arose from amino acid dissimilations by rumen microorganisms. 'Arginine was dis- similated to ornithine in all ten rumen fluid and washed cell incubations. Koreover, putrescine was found to be produced in 50 per cent of the fermentations (5 out of 10) and not to be produced in the other 50 per cent. Lysine also gave rise to cadaverine in 50 per cent of the fer- mentations (5 out of 10) but did not in the other 50 per cent. Putrescine and cadaverine when produced arose from the same inocula and were likewise absent in other sets of similar inocula. In addition, whenever these two amines were produced in rumen fluid incubations, they were like- wise produced by washed cell suspensions from this same rumen liquor. The converse of this was also true-—an ab- sence in one medium was followed by an absence in the other medium. The inocula which produced putrescine and cada- verine were obtained from two fistulated cows whereas the inocula which failed to produce these two amines came from three different fistulated animals. These results would 'indicate that inocula differences do occur in cows on si-- milar rations and are reflected in subsequent dissimilation 87 studies. Crnithine, putrescine and cadaverine were identified by the same means as were used with delta amino valeric acid. The addition of known ornithine, putrescine and cadaverine to the unknown lysine and arginine dissimila- tion mixtures did not result in the appearance of any new Spots when tested in several solvent systems. This was true using both one- and two-dimensional paper chromato- graphy. . Figure l is a photograph of a rumen fluid incubation study. Reading from left to right strips 1 and 2 are DL- lysine, before’énd after incubation, strips 5 and 4 are L-lysine, before and after incubation, and strips 5 and 6 are DL—tryptOphan, before and after incubation. Strip 7 is a post-incubation sample of arginine from a previous study. Strip 8 is a chromatogram of the rumen fluid inocu- lum prior to incubation. The lysine strips demonstrate the much greater dis- similation of the L-lysine versus the DL—lysine (lC mch./ ml. in each case) and the formation of cadaverine in both cases, being more pronounced from L-lysine. Delta amino valeric acid was produced in both cases, being slightly more apparent from L-lysine; but concentrations were too low to be apparent on the chromatograms. The arginine strip shows the dissimilation of arginine to ornithine; small quantities of delta amino valeric acid are present Figure 1. in vitro rumen fluid incubation studies with L-lysine, DL=1ysine, DL-tryptophan and L-arginine. 89 but are not visible. The diminution in spot intensity on chromatogram 6 when compared to chromatogram 5 demonstrates a partial dissimilation of DL-tryptophan. The presence of ninhydrin-reactive spots on strips 5 and 6 establish the presence of endogenous amino acids or amines in the rumen fluid. All these endogenous compounds were dissimilated in the 24 hr. incubations to a concentration where they were no longer detectable by the methods employed. The disappearance of the endogenous ninhydrin-reactive spots is demonstrated in strip 6. Casein hydrolysate and 18 amino acids were incubated in both rumen fluid and washed cell suspensions at pH 6.5. All the incubation mediums were tested for amines and only putrescine and cadaverine were found. Since most of the bacterial decarboxylases are formed in large quantities only when the organisms are grown in an acid medium, pH 2.2 to 5.5 depending on the organism (Gale, 1946), a single study was initiated wherein histidine, lysine, phenylala- nine, tryptophan and arginine were incubated at pH 4.5, 5.5 and 6.5. Phenylalanine, histidine, tryptOphan and arginine did not form amines at any of the three pH's used. Lysine did not form amines at pH 4.5 and 6.5 but a marked amount of cadaverine was produced in the rumen liquor buf- fered at pH 5.5. Paper chromatography established that ap- proximately 40 to 50 per cent of the lysine had been con- verted to cadaverine at pH 5.5 whereas only nine per cent of the lysine was completely deaminated (Table 10). The paper chromatOgraphic procedure used indicated that 100 per cent of the arginine was converted to ornithine when incubated at pH 6.5 but only 80 per cent was converted at pH 5.5.» No putrescine was produced at either pH. Sincef Sirotnak 2E.§l- (1953) reported that the presence of mal- tose in the incubation medium significantly increased the production of ammonia and carbon dioxide from aspartic acid, a trial was carried out in which arginine and lysine (lO mch./ml.) were incubated with 0.2% maltose in unbuf- fered rumen liquor. In this study the final pH of the mediums were 5.6 for the lysine flask and 5.8 for the ar- ginine flasks; cadaverine was produced whereas putrescine was not. These results from the three pH studies and the maltose addition study indicate that lysine decarboxylase activity in rumen fluid is much greater at a more acid pH than is ornithine decarboxylase. Table 10 shows the per cent dissimilation, as de- termined by ammonia production, of each of the five amino acids at pH 4.5, 5.5 and 6.5. Ammonia production was markedly reduced at pH 4.5. This pH appeared to almost completely inhibit rumen microbial activity and also to deatroy the colloidal stability of’the rumen liquor inocula. In the process of the Ethyl ether extraction of_post incubation samples and the subsequent evaporation of the ethyl ether into 2N HCl,it was noted that tryptOphan 91 Ammonia production from amino acids at three pH's in 24 hr. rumen fluid incubations (lO mch./ml. of amino acid). W Ammonia N (mg./lOO ml.) Sample pH Theor. Aetual % Dis- Yield' Yield similation L-Phenyalanine 4.5 14.01 0.00 0.00 5.5 14.01 11.02 78.65 6.5 14.01 10.62 75.80 DL-Tryptsphan 4.5. l4.0l 1.87 1.33 5.5 14.01 7.96 56.81 6.5 14.01_ 9.27 66.16 L—Lysine 4.5 27.99 0.54 1.22 5.5 27.99 2.56 9.14 6.5 27.99 6.19 21.74 L-Histidine 4.5 14.01 0.54 2.42 5.5 14.01 2.11 15.06 6.5 14.01 5.05 55.90 L—Arginine 4.5 56.04 1.24 2.21 5.5 56.04 55.16 59.20 6.5 56.04 57.44 66.80 dissimilation samples turned red in the acid solution. m L he red color did not appear, however, when distilled water re- placed the acid solution. Several attempts were made to 92 isolate the intermediary product but these attempts failed due to the low concentration present and the marked solu- bility of this compound in the various solvent systems tested. Paper chromatograms examined under ultra violet light and sprayed with Ehrlich's reagent demonstrated the presence of two compounds, indole and skatole, in rumen fluid and indole alone in washed cell suspensions. Indole gave a carmine color with Ehrlich's reagent both immedi- ately after spraying and 24 hours later, whereas skatole was carmine initially and turned blue 24 hours later. In- bdole and skatole were clearly separated and appeared as two separate spots under ultraviolet light using a solvent mixture of 10 parts isopr0pyl alcohol, 1 part ammonium hy- droxide (28% ammonia) and 1 part water. 7 Two samples of rumen fluid and two samples of washed cell suspensions all of which had been incubated with DLp tryptOphan were extracted with ethyl ether. The ethyl ether was evaporated off into 4 m1. of distilled water. The ultraviolet spectrum of these samples and indole and skatole standards are presented in Figures 2 and 5. Only one spectrum of the washed_cell suspension and the rumen fluid sample are presented in Figures 2 and 5, respectivekn since the duplicates were essentially the same. A quantitative estimation of the amount of indole and skatole formed from the in_vi§gg fermentation of tryp- tOphan was performed using both rumen fluid and-washed cell ABSORPTION 93 Indole washed Cells Illlllllv..oiOlllll:It!IIIlltlllllllltlllflllllllllll'IIIII 250 260 270 280 290 300 SIC WAVELENGTH - Mu Figure 2. Ultraviolet spectra of the tryptOphan fermentation product from washed cell suspensions and an indole standard. ABSORPTION 94 Skatole Rumen Fluid II'll.Illlllllllllllll'IlllllIll'lIIIHIIIIIIIIIIIH'I||Illlll' 250 260 270 280 290 300 3 WAVELENGTH-Mu. Figure 3. Ultraviolet spectra of the tryptOphan fermentation product from.rumen fluid and a skatole standard. 95 suspensions, with and without the addition of maltose. In- dole and skatole were estimated by the method of Meyers (1950) and the results are shown in Table 11. The produc— tion of indole and skatole was much greater in rumen fluid than in washed cell suspensions. Indole formation could be increased in washed cell suspensions by increasing the incubation period from 24 hr. to 48 hr. Maltose addition to rumen had little effect on indole and skatole formation whereas maltose addition resulted in the production of small quantities of skatole in washed cell suspensions. An estimation of the extent of formation of volatile fatty acids from certain amino acids was performed using rumen fluid with and without maltose. Rumen fluid was in- cubated with either lysine, ornithine,arginine, delta amino valeric acid or tryptOphan as the sole substrate and with each amino acid plus 0.2%lmaltose as substrates. The volatile fatty acids formed in rumen fluid alone are pre- sented in Table 12 and in rumen fluid plus maltose in Table 13. Results in rumen fluid alone were variable; ly- sine and ornithine both formed small amounts of fatty acids, whereas delta amino valeric acid, tryptOphan and arginine all decreased the volatile fatty acid concentrations to levels lower than the endogenous values. Results in rumen fluid plus maltose demonstrated that DL-tryptOphan, L— arginine and L—lysine were dissimilated to volatile fatty acids, largely acetic, whereas DL-lysine was not. Delta 96 I Table ll. Indole and skatole production in rumen fluid and washed cell dissimilation studies (l0 mcL./nl. of tryptOphan; 24 or 48 hr. dissimilation stu- dies). :1 mcg./ml. of indole and skatole Incubation Rumen Fluid . Time Indole Skatole Blank, zero hour 24 hr. 0.7 0.0 Endogenous 24 hr. 1.0 0.0 Tryptophan 24 hr. 12.8 6.0 TryptOphan 24 hr. 19.2 7.8 Tryptophan + maltose (0.2%) 24 hr. 17.6 6.9 Tryptophan + maltose (0.2%) 24 hr. 14.6 5.7 Incubation flashed Cells .Time Indole Skatole Blank, zero hour 24 hr. 0.0 0.0 Endogenous 24 hr. 0.0 0.0 Tryptophan 24 hr. 1.0 0.0 Tryptophan . 24 hr. 0.8 0.0 Tryptophan 48 hr. l.8 0.0 Tryptophan 48 hr. 1.6 0.0 Tryptophan + maltose (0.2%. 24 hr. 0.8 0.5 0.2 Tryptophan + maltose (0.2%) 24 hr. 1.0 97 .mwow canoamb osflam spawn H He.mmH m¢.mm as.ma o¢.om mm.a mo.a wa.o mm.o asassnsopuqm sm.mm 50.04 Ho.ma .sm.sa ou.m on.m mm.o om.o osaqamnmuq mm.mmH om.mm mm.m~ ma.om oo.m ne.m em.o sm.o H<> as .nnmnm oaaamm .vfisam amass ma macapmawaammflv dflom osflam mo moanspm oppfi> mm Scam mvflom hppmw mawpsao> Ho mommpsmonom Amaoa was A.HE\.AoEV macapmnpaoocoo 059 .NH manna .0400 044040> oqfiem maamm 4 00.000 00.00 00.m44 50.00 05.40 _00.0m 0m.40 00.4 40.0 00.0 40.0 40040 00.000 05.50 50.044 40.04 00.00 00.00 00.40 00.4 00.0 40.0 00.0 004004-40 00.00m 40.50 04.044 00.04 00.00 00.0w 00.00 05.4 50.0 00.0 00.0 004004-4 00.040 00.00 00.004 00.04 00.00 00.04 00.40 00.4 04.0 00.0 00.0 00404004-4 .00.040 00.00 00.004 00.04 54.40 00.04 00.40 00.4 00.4 04.0 -00.0 0000000404-40 00.00m 00.50 40.544 00.0w 00.00 00.0w 00.40 00.4 04.0 00.0 45.0 0000000000 000004 05.00 05.05 00.00 00.0m 54.04 40.04 05.4 04.0, 00.0 ,00.0 00400004 004o< o4q04m 044%» 04404 no 40000 as 040000 as -000 as :00 0s :00 02 .0040+ 040000 .0004408 am.0 + 04044 00004 04 00040044040040 0400 00400 40 0040000 0444> mm 0044 00400 04404 044404o> mo mmwmpcmo4ma 40408 0:0 A.HS\.40s0 0:04p04pqmoqoo 039 .m4 04909 amino valeric acid again lowered the total volatile fatty acid concentration to a level below that found in the endo- genous fermentation. The major effect was to lower the acetic acid concentrations, both with and without maltose, with little effect on the other individual fatty acids. Since the addition of an amino acid produced only a three to five per cent increase in the total volatile acids and this was distributed into five fractions, this analysis proved to be of limited value. An estimation of the extent of formation of volatile fatty acids from lysine, ornithine, delta amino valeric, tryptOphan, aspartic acid, arginine and Serine was per- formed using washed cell suspensions of rumen microorgan-, isms. In order to have a sufficient acid concentration to perform the volatile fa ty acid analyses, the incubation mediums were concentrated five fold. dith the exception of serine, aspartic acid and arginine, the volatile fatty acid concentrations in the amino acid dissimilation flaSks closely approximated the levels in the endogenous flasks. Differences between the endogenous and eXperimental values are presented in Table 14. .Serine and aspartic acid formed ,0 primarily acetic and prOpionic c 00 .4441 v.3rn"*\+-4‘Y"l' 'zr‘w vw. -‘ 0108, 0‘00000i.0 , nh€.03* w arginine yielded a much more uni orm mixture of volatile fatty acids. 100 00.00 55.50 05.04 40.00 00.04 00.04 05.0 04.04 00.0 50.0 00.0 0n4c4m0<14 05.00 00.44 00.04 04100 00.00 00.0 00.0 00.0 00.0 00.0 00.0.4404ns000004-4 40.00 00.05 00.00 00.5 54.0 00.04 04.0 00.0 00.0 00.0 00.0 004000-40 004o< 04s04m 0440» 04004 no 40000 00 040000 05 -000 0: -0m 04 -00 0a -0040 {I III---.l- . .00540> mzosomooqo map 03040 0044040000000 0404Im>44 0 000004004 00540> .00040 IsmmmSm 4400 000003 04 00040044840040 0400 00480 40 0040200 ouu4> a4 8004 00400 04004 044004o> Ho mommpaoopmm 40409 0cm 4.4a\.4cav 02040004nmoqoo was .34 @4909 lOl lg Vivo Studies The final phase of this thesis is concerned with the in 1212 rumen microbial dissimilations of L-arginine, L- lysine and DL-tryptophan and a comparison of the extent to which the in vitgg dissimilations were duplicated in 1119. A second aspect of this study was a qualitative examina- tion of the amino acids and amines of jugular vein blood plasma after administering these three amino acids into the rumen through a fistula. The results of these three trials are summarized in Figure 4. Arginine had the most pronounced effect upon ru- men ammonia concentrations. Rumen ammonia levels continued to rise for at least the first six hours after administra- tion of arginine but had decreased by ten hours. L-lysine had little effect upon rumen ammonia levels the first three hours after administration, but thereafter L-lysine tended to keep rumen ammonia concentrations at much higher levels than the endogenous ration. The ammonia levels after ly- sine administration, however, were still considerably below those from arginine supplementation. The addition of DL- tryptOphan had little effect upon rumen ammonia concentra- tion until about six hours after administration. Even then these levels were much lower than those noted for L- arginine and L-lysine. By the fourteenth hour the trypto- phan supplemented and endogenous rumen levels were the same. 102 Figure 4. The levels of ammonia nitrogen in rumen liquor after the administration of three amino acids \ I ' ° 3’ ’ .. . °. ' 3 “ \ '. I, ' \ 3 I ' : ”8 . I C O O O \ I Z ‘ §\» / 3: 4’5! 1 \\. I ‘n O. 1 , Do 4 Z . \ I 05.“! O zazlfl cog- 3’5; '2 /‘\\ Egg! iii; ‘ :5 O 4 \ 2:] . 2 "39 \‘ uo.J< ‘ Z \ ° - . \ -05 ‘ .. \ \ ( o. \ 1 c .,' g \ '99:, . ’I h. ” . '( I, ‘ j> **"=; , 0 8 z / I ‘ I; /. '00 I .l’ ‘,a"" ‘ ’1’ / .' f . r iv o J - ' I 0” 2"! I, an“ "E' ””0“ ‘ A v r VJ. r r I v v v v v O N o o o v in o o o v N o ' ' M M N N N N N - - - - - o o v N uonon Nawna swoon sad N-‘HN 'ow 105 The in vivo studies with L-arginine, L—lysine an (2. DL-tryptophan substantiate the in ZEEEQ studies with these three amino acids. In both mediums, L-arginine markedlv raised the ammonia concentration in the rumen fluid over the endogenous levels. L-lysihe and DL-tryptOphan likewise were dissimilated at similar rates in 111g and in vitro. L-lysine was one of the intermediate amino acids in dissi— milation rate in gigg wlereas DL—tryptOphan was only slowLy dissimilated in gizg. These results were similar to those obtained in vitro; each amino acid falling, in zixg and in vitro, into one of three general classes with regard to their magnitude of dissimilation. At the same time samples were being prepared for am- monia analyses, rumen liquor samples were likewise prepared for paper chromatographic analyses to indicate and identifly the degradation products of the amino acids. AIPHTUMICETflo) glutamic acid (g.a.), alanine (al.) and delta amino valeric acid (d.a.v.a.) were present in the rumen liquor immedi- ately after the addition of L-arginine to the rumen. The ruminal concentrations of these amino acids changed as a function of time in the followi g manner: 1 hr. arg. decreased; ornithine (orn.) was formed; 5.a., al., d.a.v.a. increased. 2 hr. arg. decreased; g.a., al., orn., d.a.v.a. in- creased. }, 4, 6 hr. arg., 31., increased. 10 hr. arg., al., g.a., orn. decreased; d.a.v.a. in- creased. 14, 25 hr. only trace amounts of all five amino acids present. g.a. decreased; crn., u.a.:.a. 104 Equal amounts of arginine and ornithine were present on the chromatOgram of the sample taken at the sixth hour. Lysine (1y.), glutamic acid (g.a.), alanine (al.) and delta amino valeric acid (d.a.v.a.) were present in the rumen liquor immediately after the addition of L-lysine to the rumen. The ruminal concentration of these amino acids changed as a function of time in the following manner: 1 hr. 1y. decreased; al., g.a., d.a.v.a. increased. 2 hr. 1y. decreased; d.a.v.a. increased; al., g.a. remained the same. 3 hr. ly., al. decreased; d.a.v.a. increased; g.a. remained the same. 4, 6, 10 hr. ly., al. decreased; d.a.v.a., g.a. re- mained the same. 14, 25 hr. ly., al. trace; d.a.v.a., g.a. remained the same. ' The lysine concentration decreased to about 50 to 60 per cent of the original level on the chromatogram of the sampha taken at the sixth hour. Alanine and glutamic acid were present in low con- centrations in both studies; the largest increases being one hour after lysine administration and two hours after arginine addition. Cadaverine and putrescine were not pre- sent in detectable concentrations in either of these ig vivo dissimilation studies. The chromatOgrams of the rumen fluid from in vivo dissimilations of L-arginine and L- lysine are found in Figures 5 and 6, respectively. The chromatograms of the ip vivo dissimilation of DL-tryptoPhan failed to reveal the formation of new nin- hydrin-reactive products. Glutamic acid and alanine were 105 Figure 5. Paper chromatograms of rumen fluid from in vivo studies with L-arginine. ‘1' Paper chromatograms of rumen fluid from in vivo studies with L-lysine. Figure 6. 107 again present but decreased in concentration more rapidly than with the lysine and arginine studies. TryptOphan con- centrations decreased only slightly during the first three hours and then a gradual decrease occurred until by the tenth hour the tryptOphan concentration was markedly re- duced. The chromatograms of the samples taken at 14 and 24 hours showed only small amounts of tryptOphan. Indole and skatole determinations were made on the samples that had been preserved for the ammonia analyses. These results are found in Table 15. The indole concentration did not Table 15. Indole and skatole production from in vivo dissimilation studies with DL-tryptophant- n1c7,/ml. of indole and skatole. Endogenous . I: fii-TryptOphan Time Indole Skatole Indole Skatole 0 hr. 0.4 O 0.5 0.0 1 hr. 1.0 O 0.9 0.0 2 hr. 0.8 O 1.6 0.0 5 hr. 1.2 O 1.8 i 0.0 4 hr. 1.2 ' O 2.2 0.0 6 hr. 0.9 ‘ 'O 5.6 ‘ 0.0 8 hr. 0.9 O 4.8 3.6 10 hr. 0.4 O 4.2 . .2.0 lBkHu 0.2 O 1.5 0.0 24hr. 0.3 O 0.4 0.0 108 increase above the control until two hours after trypto- phan administration; then it showed a steady increase up to 4.8 (AC f./m1. eight hours after the administration of DL-tryptOphan. This maximum was followed by a steady de- cline in indole concentration. Skatole was detected only in the eight and ten hour samples which correspond to the times of highest indole concentrations. The individual amino acids in blood plasma from the jugular vein were identified on two-dimensional paper chromatOgrams by their Rf values. Other methods used were the color reactions of each amino acid when sprayed with the cupric nitrate-ninhydrin reagent of Moffat and Lytle (1959), certain individual tests-~proline (isatin), argi- nine (Sakaguichi) and citrulline (Ehrlich's) and compari- son with the chromatograms of bovine plasma amino acids by CoulSon 22 gl. (1999). These chromatograms were similar to those of walker (1952) and Coulson 22 gl. (1959) and failed to reveal the presence of tryptOphan in the plasma. Gordon (1949), however, has reported the presence of tryp- tophan in bovine serum. Since the plasma amino acid sam- ples in this treatise were prepared by a method similar to that of walker (1952) and Coulson gt gl. (1959), it is possible that tryptOphan was lost during the extraction. TryptOphan was also absent from the plasma amino acid chromatOgrams of the tryptOphan-supplemented animal. ' 109 The qualitative estimation of amino acids in blood plasma after intraruminal administration of L-lysine, L— arginine and DL—tryptOphan are summarized in Tables 16, 17 and 18, respectively. The lysine and tryptOphan studies were on one animal whereas the arginine study was on an- other. These results are based upon the visual qualita- tive examination of paper chromatograms of plasma amino acids prepared from jugular blood prior to the administra- tion of the amino acid and one, two and four hours follow- ing the amino acid addition to the rumen. Previous studies in this laboratory, involving visual comparisons of paper ,chromatograms, had indicated little effect on the endogen- ous levels of the amino acids from the blood plasma of the jugular vein through the first four hours following the first two hours after feeding of the experimental ra- tion. 9 These results were difficult to interpret due to the limited number of analyses and the poor sensitivity of the analytical technique. The two most striking observa- tions, however, are the lack of effect of tryptOphan sup- plementation on other plasma amino acids and the marked increase in lysine concentration of venous plasma at four hours after the administration of L-lysine to the rumen. [Both L—lysine and L-arginine supplementation increased the concentration of several plasma amino acids within one hour following the additions of each amino acid to the rumen. 110 Table 16. The relative effects on jugular plasma amino acid concentrations of L-lysine addition to the rumen. Intensity relative to Relative the 0 hr. sample at Amino Acid -Intensity 1 hr. 2 hr. 4 hr. Aspartic Acid Very Weak -1 — _ Glutamic Acid Strong +3 82 S Serine Strong + S S Glycine Strong 8 S Threonine Weak ++4 + + Citrulline Moderate + S S Glutamine Moderate + S S Alanine Very Strong + + Tyrosine Jeak + + Valine Strong ++ + + Methionine Moderate ++ + + Leucine Moderate ++ + + Phenylalanine Moderate ++ + - + Proline Weak - - - Unknown Ju'eak S + + Histidine deak - _ - Lysine Moderate S - ++ Arginine Moderate S - - AABA5 fleak S S - 2 S 2 equal to. 5 4 ++ = more than +. + = more than. 5 Alpha amino butyric acid. Table 17. 111 The relative effects on jugular plasma amino acid concentrations of L-arginine addition to the rumen. W Intensity relative to Relative _£he 0 hr. sample at Amino Acid Intensity 1 hr. 2 hr. 4 hr. Aspartic Acid Very weak 82 s s Glutamic Acid Strong +5 s s Serine Strong + + S Glycine Strong + + + Threonine Weak + S S Citrulline Moderate + + + Glutamine Moderate + + + Alanine Very Strong + S S Tyrosine Weak S S S Valine Strong + + + Methionine Moderate + S S Leucine Moderate + S S Phenylalanine Moderate S ‘1. S Proline Weak S S S Unknown Weak S S S Histidine Weak S S S Lysine Moderate + + + Arginine Moderate + + + AABA4 Weak s s s - a less than. 2 S 3 equal to. 5 4 + a more than. Alpha amino butyric acid. Table 18. 112 -- -r_.______.____-. ~_.___.._.____.,, Vevflwm_fl_ _.___.____..._.'——_.—_ w——~—.——.—‘_—- Intensity relative to The relative effects on jugular plasma amino acid concentrations of DL-tryptophan addition to the rumen. the 0 hr. sample at Relative Amino Acid Intensity 1 hr. 2 hr. 4 hr. Aspartic Acid Very Weak S2 -l - Glutamic Acid Strong S S S Serine Strong S S S Glycine Strong S S S Threonine Weak S S S Citrulline Moderate S S S Glutamine Moderate S S S Alanine Very Strong S S S Tyrosine Weak Q S S S Valine' Strong S S S Methionine Moderate S S S Leucine Moderate S S S Phenylalanine Moderate S S . S Proline Weak S S - Unknown Weak S S S Histidine Weak S S S Lysine Moderate S S .S Arginine Moderate S S S; AABA3 Weak s s s - a less than. 5Alpha amino butyric acid. 115 Plasma aspartic acid, proline, histidine and alpha amino butyric acid, however, failed to show a detectable increase. The addition of L—arginine to the rumen resulted in only small increases in arginine on the chromatOgrams. Venous serum levels of indole and skatole were less than 0.25 mtg . per ml. at zero, one, two and four hours following the addition of DL—tryptophan to the rumen. These samples, however, were taken prior to the maximum concentrations of indole and skatole in the rumen. DISCUSSION Since ammonia is formed in the latter stages of pro- tein catabolism and is the prime nitrogen source in pro- tein anabolism, it is necessary to understand the condi- tions under which ammonia is released from amino acids. This study was designed for such a purpose. The experiments 1g zixg using dry cows fitted with permanent rumen fistulas and ip zgpgg using rumen liquor and washed suspensions of rumen microorganisms have shown that amino acids are attacked individually by rumen micro- organisms. Since the rate of attack upon single amino acids was considerably more rapid and extensive ip 3112 and in rumen liquor than when washed cells were used, it appeared that the method of preparation of the suspension somehow inactivated the enzymes responsible for the deami- nation. A second possibility for decreased deamination could have been a selection of the non-deaminating popula- tion in the washed cell preparations or a lysis or death of the deaminating microorganisms. The first supposition appeared more likely and was the one investigated. How- ever, the individual usage of 48 hr. enriched cultures, pyridoxamine, pyridoxal phosphate, magnesium ion, all p0— tassium buffer, methylene blue or catalase failed to sig- nificantly promote ammonia production over the control values and were still low as compared to rumen liquor and 114 115 in 1339 ammonia production. Lewis (1955) obtained in- creases in the activity of washed suspensions of rumen microorganisms by using greater care in the maintenance of anaerobiosis, by the addition to the medium of a portion of ammonia-free, Seitz-filtered rumen contents and by the use of a thicker suspension with phosphate buffer at pH 7 containing 0.02% (w/v) glutathione. However, these stimu- lations were slight and conditions must have differed sig- nificantly from those present in rumen liquor. Increasing the number of amino acids to three or four in the suspen- sions failed to produce greater ammonia levels as might be expected if a Stickland type of reaction predominated. An increased rate of production was obtained, however, in one study in which alanine and proline were incubated together. The ig xipgg studies demonstrated that amino acids may be divided into three groups with regards to their re- lative rates of dissimilation. Arginine, aspartic acid, serine, threonine and cysteine were attacked most com- pletely, followed by glutamic acid, phenylalanine, lysine and cystine forming an intermediate group and a third group in which deamination was much less pronounced was tryptOphan, delta-amino valeric acid, methionine, alanine, valine, isoleucine, leucine, ornithine, histidine, glycine, proline and hydroxyproline. These findings agree qualita- tively with the results of Sirotnak 22 Q1. (1955) and Lewis (1955) with a few exceptions, notably arginine, 116 lysine and tryptOphan. Both of these authors reported that washed cell suspensions deaminate aspartic acid at a much greater rate than arginine whereas rumen liquor incu- bated for eight hours in the present investigation demon- strated that arginine contributed approximately five times as much total ammonical nitrogen as did aspartic acid. A marked increase in rumen ammonia levels was also noted when arginine was employed in the 11 1119 studies. It must be remembered, however, that the 11 1119 and 11 1113p rumen fluid studies were not limited to the rumen bacteria but also included the rumen protozoa. Since Barrentine 21 Q1. (1957) and Emery g1 31. (1958) obtained practical control of bloat with penicillin, a trial was designed to study the effects of penicillin on amino acid dissimilations. Three I. U. of penicillin per ml., the approximate ruminal levels in the study by Emery 31 a1. (1958), had no apparent effect on amino acid cata- bolism whereas 50 I. U. per ml. markedly inhibited amino acid dissimilation. The experiment with serine would indicate that spe- cific materials interfering with endogenous metabolism may be useful tools in amino acid dissimilation studies since the addition of arsenate and fluoride addition to the medium increased ammonia production over the control values in eight hour washed cell incubations. 117 Since delta-amino valeric acid has been shown to be a product from the dissimilation of arginine, lysine, orn- ithine and proline, it would appear that this amino acid may be an important intermediate in amino acid metabolism by rumen microorganisms. Furthermore, delta-amino valeric acid was found to be present in six of eleven samples of rumen fluid taken two to four hours after feeding. Delta- amino valeric acid arose in arginine, lysine and ornithine fermentations in the absence of any other added amino acid whereas proline appeared to give rise to this amino acid only in the presence of added alanine. Dehority g1 g1. (1958), however, reported that proline may be transformed into delta-amino valeric acid in the absence of another amino acid. The fact that this amino acid may be formed in the abSence of other added amino acid does not exclude its formation via the Stickland reaction. Hydrogen dona- tors may be formed in the lg 11139 and 11 1119 studies in the course of carbohydrate metabolism, from amino acid syn— thesis, or by the autolysis of the rumen microorganisms themselves. Both ornithine and arginine were reported by Woods (1956) to be good hydrogen acceptors in amino acid fermen- tations by 91. sporogenes. Arginine did not appear to act by first being converted to ornithine in the studies by floods due to the fact that ammonia levels were higher in the reaction flasks containing pairs of amino acids than 118 could be accounted for as an ornithine reduction. The urea formed in such a reaction was not degraded by Q1. Sporogenes. In the studies reported here delta-amino va- leric acid accumulation closely approximated the formation and disappearance of ornithine. Such findings would indi- cate that at least part of the delta-amino valeric acid formed in arginine dissimilation studies arose from orni- thine. The metabolic pathways by which lysine could yield delta-amino valeric acid are few. Most likely, it appears, is the conversion of lysine to an alpha-keto—delta-amino derivative, which through oxidative decarboxylation would yield delta-aminovalerate. The fate of delta-amino valeric acid in the rumi- nant is still unknown. One study by Dehority g1 g1. (1958), employing uniformly labeled 014 proline, found that the major portion of the non-amino acid activity was pre~ sent in the volatile fatty acids, particularly the C5 fraction. By increasing the washed cell incubation time from 24 hr. to 50 hr., a decrease was found in the C5 fraction with a subsequent increase in the C5 fraction. An analysis of the C5 fraction revealed valeric acid and an unknown component.' The concentration of this unknown component was five times greater at 50 hr. than at 24 hr. When this isolated unknown material was made alkaline, it could be extracted into ether, indicating its non-fatty acid nature. An organism belonging to the genus 119 Clostridium has recently been isolated from sewage by Hardman and Stadtman (1960). The over-all reaction cata- lyzed by this organism was a coupled oxidation reduction process in which two moles of delta-amino valerate were converted to two moles of ammonia and one mole each of valeric, propionic and acetic acids. An intermediate in this dissimilation appeared to be 5 keto-valeric acid and may be the unknown component reported by Dehority 31 Q1. (1958)- There is very limited information in the literature on the production, absorption and excretion of indole in the normal ruminant. Spisni and Cappa (1954) reported that indole was present in rumen contents of 15 cattle just after slaughter; the amount ranged from 0.09 to 5 mg. per liter. This figure agrees with the levels reported in this study. Indole formation in the rumen has not pre- viously been demonstrated to be due to bacterial action on . tryptOphan, though it has in monogastric animals (Peterson and Strong, 1955). The high levels of indole formed from the 11 11119 and 1Q 1119 fermentation of tryptOphan very strongly suggest this possibility. Indole production has been demonstrated in pure cultures of rumen bacteria (Gutierrez, 1955; Blackburn and Hobson, 1960a). The mech- anism of the over-all reaction of indole formation was established by floods 21 11. (1947). Employing partially purified tryptOphanase preparations from extracts of 120 Escherichia gg11, it was demonstrated that the reaCtion yielded indole, pyruvic acid and ammonia in approximately equimolar ratios, and that there was no uptake of oxygen. When a goat was changed from an alfalfa to a Lepidium diet, the indole in the blood rose from zero to 1.0 p.p.m. and this high level at the time of milking caused a high concentration in the milk fat (Conochie, 1955). Conochie (1955) also reported that when indole was given by mouth to a goat three-fourths was excreted in the urine as the indoxyl etheral sulfate. Indoxyl substances were estimated in the urine of 78 cattle by Spisni and Cappa (1954); the amount excreted ranged rom 0.01 to 0.5 g. per 100 ml. urine. Nhen the proportion of grasses in the hay was high, the amount of indoxyl substances in the urine was relatively low, and it increased when the pro— portion of leguminous plants was high. One can conclude that the higher indole levels on leguminous rations were due to the higher tryptOphan content of these plants andfln? the greater availability of this tryptOphan to the rumen microorganisms. The results of this study indicated that, in some instances, cadaverine and putrescine may be produced by rumen microorganisms.' Putrescine has been reported in a few instances in the rumen liquor of goats and sheep fed on ladino clover or pasture (Shinozaki, 1957). The pharma- cology of these two amines has been reviewed by Guggenheim 121 (1940). A short resume of his findings will be discussed. The occurrence of a second amino group reduces extensively the pharmacological action of alkylamines. Toxic oral dosages are 1.6 g./kg. of putrescine in rabbits and more than 1.7 g./kg. of cadaverine in the dog whereas subcuta- neous dosages of either amine of 0.1 g./kg. are toxic to rabbits. The chief site of action of a toxic dose of di- amines is the central nervous system and the symptoms are motor paralysis, convulsions, dyspnoic breathing, retarda- tion and arrhythmia of the pulse, and a lowering of the blood pressure and temperature. Rather convincing evidence now exists which shows that the pattern of amino acids in the diet markedly in- fluences the level of some free amino acids in the blood. In poultry, Charkey g1 g1. (1955) and Almquist (1954) ob- served good correlations between amino acid levels in chick blood and composition of dietary protein. Denton and Elvejehm (1954a, b) reported that the portal and radmd vein concentrations of individual essential amino acids in dogs were rapidly increased in prOportion to the levels supplied by the test proteins, casein and beef. In the case of the imbalanced protein, zein, which lacks lysine and tryptOphan, lysine levels were depressed whereas tryp- tophan concentrations were well maintained on the zein diet. 122 The results of the 1Q 1119 investigations definitely show that individual amino acids differ in their rates of deamination in the rumen. Each of the three amino acids studied--L-arginine, L-lysine and DL-tryptophan--however, had a different effect on rumen ammonia levels. Although the plasma amino acids from the jugular vein were studied only qualitatively, certain trends were notable. The more readily dissimilated amino acids, arginine and lysine, had a positive effect on plasma amino acids whereas tryptOphan had very little effect through the first four hours follow— ing the amino acid administration. Furthermore, this ef- fect was evident in several of the plasma amino acids, in- cluding the essential amino acids; it was no more marked in the amino acid administered except in the lysine con- centration of the plasma amino acids taken four hours after lysine administration. The latter phenomenon may have been due to intestinal absorption of the lysine. Although orni- thine was present in readily detectable amounts in rumen liquor as early as one hour following arginine administra- tion to the rumen, ornithine was absent on the chromato— grams of the blood plasma taken one, two and four hours after the administration of arginine. These results would indicate that the amino acid administered was either not being absorbed from the rumen, or if absorbed, was being modified before it reached the peripheral circulation. Both Annison (1956) and Tsuda 123 (l956b) have reported evidence indicating amino acids are not absorbed from the rumen in appreciable quantities. If the blood samples had been taken at longer intervals after feeding, the effects of the inteStinal absorption of these amino acids may have been evident. The relative rates of disappearance of these amino acids from the rumen indicated that the half time of ruminal disappearance of amino acid supplements to rations would be in the order of six to eight hours. SUNJARY The amino acids serine, aspartic acid, glutamic acid, arginine, lysine, cysteine, cystine, threonine and phenylalanine were readily dissimilated when added to incu- bating rumen fluid or washed suspensions of rumen micro— organisms. Tryptophan, histidine, methionine, ornithine, valine, alanine, leucine, isoleucine, delta-amino valeric acid, glycine, hydroxyproline and proline were dissimilatal at lesser rates. The dissimilation rates were more rapid and complete in rumen fluid studies than in washed cell suspension. Three or four amino acids incubated together differed from the summation of the ammonia formed from each amino acid only in the cases where proline and ala- nine were incubated together. The individual usage of 48 hour enriched cultures, pyridoxamine, pyridoxal phosphate, magnesium ions, all potassium buffers, methylene blue or catalase in washed cell incubations failed significantly to promote ammonia levels over the control values and were still lows as compared to rumen liquor and in ElXQ ammonia production. Ammonia production and amino acid disappear- ance, as noted by paper chromatOgraphy, were closely cor- related. The D- and L- forms of tryptOphan were both dissi- milated. TryptOphan yielded indole and skatole. Arginine yielded ornithine, putrescine and delta—amino valeric acid. 124 125 Lysine yielded cadaverine and delta-amino valeric acid. Penicillin at 5 I.U./ml. did not inhibit any of the dissi- milations whereas 50 I.U. caused a marked inhibition. Tests for amine production from casein hydrolyzate and in- dividual amino acids at pH 4.5, 5.5 and 6.5 were negative, except for cadaverine and putrescine. Arginine produced the highest levels of ammonia in eight hour rumen liquor incubations. The presence of arsenate or fluoride in- creased ammonia production from serine over the control value. Glutaric acid was not dissimilated in zitgg by ru- men microorganisms. Another phase of this study was concerned with the in 3119 rumen microbial dissimilation of L-arginine, L- lysine and DL-tryptOphan and a comparison of the extent to which the in zitgg dissimilations were duplicated in xizg. The in zixg dissimilations were in good agreement with the in gitgg studies. Arginine and lysine both pro- duced delta-amino valeric acid. Arginine also yielded ornithine. Indole and skatole were formed from tryptophan. Rumen ammonia levels in zigg paralleled what would have been expected from the in EEEEQ studies. 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