AMINO ACID STUDIES IN . RU-MINANTS g , INVESTIGATION OF A METHOD FOR DETERMINING THE‘LIMITING AMINO ACID f ~ - A Dissertation for the Degree of Ph. D. _ ; MICHIGAN STATE UNIVERSITY EMERSON LUCINE POTTER 1970 mn- L I B R A R Y . Michigan State University thSIS This is to certify that the thesis entitled AMINO ACID STUDIES IN RUMINANTS: INVESTIGATION OF A METHOD FOR DETERMINING THE LIMITING AMINO ACID presented by Emerson Lucine Potter has been accepted towards fulfillment of the requirements for Ph . D . degree in Animal Husbandry (Nutrition) %&7 ‘ Major professor Date October 29, 1970 0-7639 ABSTRACT AMINO ACID STUDIES IN RUHINANTS INVESTIGATION OF A METHOD FOR DETERMINING THE IMTING AMINO ACID by Fherson Lucine Potter This dissertation is concerned with developing a procedure which would identify the limiting amino acid of ruminants from plasma amino acid (PAA) concentrations. The limiting a-ino acid in nonogastric aninls is defined as the essential amino acid in the dietary protein which is least able to fulfill the animal's requirements. In compar- ison, the limiting alino acid of rulinants is the essential alino acid in the protein passing to the small intestine which is least able to fulfill the animal's requirements. Dietary protein degradation and protein synthesis by the nicrobes in the runen account for this differ- ence. Glucose infusions into the carotid artery caused decreases in the PAA levels which suggested that protein synthesis occured (Potter 33; .a_1_. 1968). If glucose stimlates protein synthesis then the amino acid limiting protein synthesis should be the plasna essentlnl nine acid whose post-glucose concentration is decreased the nost, when amassed as a percent of the pro-glucose PAA level. Potter _e_t_ 1.];- (1968) eXpressed post-glucose PAA concentrations as a percent of pre- gl'acose PAA concentrations and termed these ratios plans amino acid inlices (PAAI). Emerson Lucine Potter PHI-$1100 When A I post-glucose PAA concentration B 8 pre-glucose PAA concentration The lowest an: should correspond to the limiting anino acid since this corresponds to the PAA which was utilised in protein synthesis to the largest degree in respect to the pre-glucose level. Experinent one, a 3 x 3 htin square replicated 4 tines, was designed to investigate PAA levels of sheep as they changed due to intravenous infusions of saline and glucose (two levels of glucose, low and high). The one hour post-glucose infusion PAA concentrations were significantly (P < .05) decreased below the pro-glucose PAA levels. The pattern of decrease in please essential amino acids was significantly correlated to the amino acid cuposition of striated muscle of lanb (P < .05). Iseleucine and leucine had the lowest PAAI and, based on the assunptions outlined above. were thus indicated as the liniting amino acids. kperinent two, using 12 rate per dietary protein (egg albumin, casein. soy and sein), was designed to deter-ins the accuracy of the PAAI procedure in predicting the limiting anino acid. The PAAI proced- ure did not identify the liliting anino acid in 3 of the 4 preteins. Expressing PAA concentrations from rats (la/diet) fed test diets in experiment 3 as a percent of the average PAA concentration from rats fed whole egg, whole egg plus amino acids and casein diets (high quality proteins) , correctly identified the limiting anino acid in 6 of the 8 diets (indicated by lowest percent value). The percent values calcula- ted in this way were defined as “plasma anino acid reference indices" whereas the PAA concentrations in the rats fed whole egg, whele egg plus amino acid and casein were teraed ”reference concentrations. " Emerson Luclne Potter Plasma amino acid reference indices 2% x 100 When A = PAA concentration from rats fed a test diet B = Reference concentration from rats fed high quality proteins Plasma amino acid concentrations were determined in sheep (surgi- cally prepared with duodenal re-entrant cannulas) duodenaILly infused with proteins of ”known“ amino acid compo sition. PAAI did not identify the uniting amino acid of the infused proteins. Reference indices. calculated using the average PAA concentrations frees sheep infused with whole egg and casein as reference concentrations. identified the limit- ing amino acid in 6 of the 8 infusions. When these reference concentra- tions were caspared to PM concentrations of sheep in other studies, methionine, phenylalanine, lysine and isoleucine were indicated as the liniting amino acids. The final experiment showed that amino acid levels in blood collect- ed frcas the jugular vein and carotid artery of sheep did not differ. This suggests that blood from either site can be used in PAA studies. AHINO ACID s'mmm IN RUNINANTSs INVESTIGATION OF A METHOD Fat DETERMINING THE LIMITING AMINO ACID by Eherson Lucine Potter A DISSERTATION Submitted to Michigan State Universiw in partial fulfinnent of the requirements for the degree of Doc'rcn OF PHILOSOPHY Department of Aninal Husbandry 1970 will. an: All, rimv August 10, 19’43 Born - Owosso, Michigan 1961-196 5 . . . Attended Michigan State University B.S. Degree in Animal Husbandry 1965-1967 . . . Attended Ohio State University M.S. Degree in Aninl Science 1967-1970 . . . Attended Michigan State University Graduate Assistant PUBLICATIONS Potter, E. I... D. B. Purser and J. M. Cline. 1968. ”Effect of Various Energy Sources Upon Plasma Free Amino Acids in Ruminants.” Journal of Nutrition, 953655 FIELD OF STUDY Major Field: Ruminant Nutrition ACKNCNLEDGEMENTS I would like to express my gratitude to all persons whose efforts helped no obtain w degree. Dr. D. B. Purser is the person most responsible for guiding no in this research. Through consultations with him 11w understanding and appreciation of the problems in and possible approaches to the prob- lus in ruminant nutrition has increased. Dr. W. G. Bergen and 1h. D. E. Ullrey have given assistance through their consultations, in laboratory analysis. in interpretating the data and writing this unuscript. Dr. Thomas Adana. Rwsiology: Dr. Steve Aust, Biochenistry and Dr. H. J. Thusas. hiry. were helpful in making suggestions which increased the clarity of the manuscript and which furthered the inter- pretation of the data. Dr. W. T. Magoo was helpful during statistical analysis of the data. Paul Hassinger and Elaine Fink were of technical assistance during this research. It parents. Mr. and Mrs. Lucine Potter, have given ne continual encouragonent. lb wife . Sharon. has been very understanding and help- ful in preparation of the manuscript. ii TABLE OF CONTENTS LISTOFTABLES LIST (F APPENDIX ABBREVIATIONS I. INTRONCTION II. LITERATURE REVIEH III. IV. V. VII. VIII. Amino Acid Supply to the Runinsnt Methods of Determining the Liniting Amino Acid Plasma Amino Acids in Ruminants METHODS AND MATERIALS Blood Samples for Aline Acid Analysis kporinent (he hperinont Two Experinent Three uporinent Four hperiaent Five Statistical Analysis RESULTS hperinont (he hperinent Two Experiment Three Experiment Four Experiment Five GENERAL DISCUSSION C(NCIIJSIONS BIBLICERAPHI mu 3’ race e e 2 sh 19 19 19 20 2“ 2h 30 30 33 #5 51 69 76 78 88 Table CDVO\U|-F'UN 10 13 11+ 15 16 17 LIST OFTABLES Ekporinental Design for mperinent One Ration, Experiment One Diets, Experiment 'IVo Ezperinental Diets, Experiment Three Protein Mineral Infusion Mixtures, Emporiment Four Trace Mineral Mixture, Experiment Four Ration. Experiment Five Mean Plssna Amino Acid Concentrations by Treatment and Sample Time for Experiment (he Mean Plasna Amino Acid Indices by Treatment Experiment One Differences Botwoen the Pre-‘I‘reat-ent (To) and Post-Treatment (T1) PAA Concentrations Mean Plasm Amino Acid Concentrations of Rats Fed Differmt Protein Diets, Emperinent Two Mean Concentrations of PAA According to Saline or Glucose Treatment and Overall PAAI, Earperinont Two PAAI for Treatments in Experiment Two PAAI Divided by Egg Albumin PAAI Exporinent Tee Mean Concentration of Plasma Aline Acids of Rats Fed Different Protein Diets, EXporinent Three Mean Concentrations of Plum. Amino Acids According to Saline or Glucose Treatment and Overall PAAI, Eatperinent Three PAAI for Dietary Treatment Groups, Exporinent Three II Ii"? 21 22 23 25 28 29 32 35 37 39 1+1 1+2 Table 19 20 21 22 .23 21} 25 26 27 31 LIST OF TABLES (cont.) PAAI fro. Rats Fed Other Diets as a Percent of PAAI from Rats Fed Whole Egg er Casein Diets Plane Aline Acid Reference Indices for Ehch Dietary Treatment Group, Experiment Three Plasn Amino Acid Reference Indices Deternined for Other Experiments Using Reference PAA Concentration in Table 20 Mean PAA Concentration for Sheep modenally Infused with Different Proteins, Ihy Four Pro-Treatment. Experiment Four Mean PAA Concentration for Sheep Duodenally Infused with Different Proteins, my Four Post-Troatnent. Experinont Four Mean PAA Concentration for Sheep Duodenally Infused with Different Proteins, Day Six Pre- Treatnent. Experinent Four Mean PAA Concentration for Sheep Duedenally Infused with Different Proteins, Day Six Post-Treataent. Experiment Four Plasma brine Acid Indices for EIperinent Four PAA Referenced Indices for Ehch Pmtein Infusion Treat- ment in Experiment Four Theoretical Limiting Amino Acid of Proteins PAA Reference Indices of Conventional Sheep Mean Concentration of Venous and Arterial Plasma Anino Acids Before and (he Hour After Treatment, uperinent Five Mean Concentration of Plasma Aline Acid by Treatnent Before and (he Hour After Treatment, Ikporinont Five Mean Elam Anino Acid Indices and he-Treatnent to Post-Treatment Change in Concentration by Treatnent, Experiment Five III 50 52 53 55 57 59 61 62 63 67 LIST OF APPENDIX TABLES Appendix I Sample Calculation II Mean PAA Concentration of Saline and Glucose Treated Rats for Each Dietary Treatment, Experinent Two III Mean Concentration of Plasma Essential Amino Acids for Saline Treated Rats by Dietary Treatment, Entperimont Three IV Plane Aline Acid Concentrations of Sheep Diodenally Infused with Different Proteins, Average of [hys Four and Six, Pro-Treatment Experiment Four Plans Anino Acid Concentrations of Sheep Duodenally Infused with Different Proteins, Average of Days Four and Six, Post-Treatment Emperiaent Four IV Ii" 88 89 91 92 93 Net ABBREVIATIONS Alanine Arginine Aspartic acid Casein Corn Gluten Meal Cystine Egg Alba-in Essential Amino Acids feet Glucose or 1 hr Post Infusion Glutanic acid Glycine gran gravity Histidino hour Isoleucine kilogram Leucine liter lysine Methionine Soy-I-AA Soy+Met Val W/V WE-l-AA ABBREVIATIONS (cont'd) nillJJiter Nitrogen Ornithine Pherwlalanine Plasna Amino Acids Plans Amino Acid Indices Probability less than 5i Proline Saline or Pro-Infusion Serine Sodiun Chloride Soybean Meal in Mat 3 Praaosoy in Etporinent 4 Soybean Baal plus Aline Acids Soybean plus Methionine Threonine Total Essential Aline Acids Total Non-essential Aline Acids Tyrosine Valine Weight per Volune Whole Egg Whole Egg plus Amino Acids INTRODUCTION Ruminants are important to nan because they produce neat, milk and fiber from forages and non-protein nitrogen. Increased productive efficiency nay result from studies defining the amino acid which linits the synthesis of silk or nuscle proteins. Since the amino acid supply to the ruminant consists of a mixture of undegradod dietary and rumin- ally synthesised nicrobial proteins, the limiting amino acids of these protein. mixtures will detornine productive functions. The liniting amino acid is that acid whose availability is such that it is least able to ful- fill the aninls requirements (Al-quiet, 1951*). Since the amino acid composition of the dietary protein differs from that supplied to the shell intestine, growth responses to changes in the dietary amino acid supply will not define the liniting amino acid in runinants. Plasma amino acids (PAA) concentrations should reflect the supply of anino acids to tissues of ruminants and my be useful for identification of the limit- ing amino acid. Purser _e_t_ 33:. (1966) infused a starch-glucose mixture into the runen of sheep and reported subsequent decreases in PAA levels. When compared to the pro-infusion PAA levels, lysine was decreased the nest by this starch-glucose infusion in defaunstod sheep whereas no single amino acid was decreased more than an of the others in faunated sheep fed the sage diet. Lysine was expected as the limiting anino acid since these sheep were fed a corn diet which is liniting in lysine. It appeared that the starch-glucose induced changes in PAA levels night 1 2 b“ “”m in d°t°rl311138 “10 limiting amino acid in ruminants. The infusions of glucose into the carotid artery of sheep decreased the essential PAA levels (Potter .93. a_l_., 1968). Decreases in the absolute concentrations of each essential amino acid in the plasma were signi- ficantly (P < .05) correlated with the essential amino acid composition of striated muscle of lamb. suggesting that glucose induced protein synthesis. Theoretically. the limiting amino acid is that acid which decreased the most in respect to the amount present in the plasma before the glucose infusion; by this definition isoleucino was the limiting amino acid. Research presented in this dissertation deals with the effect of intravenous glucose infusions on PAA concentrations and the determina- tion of the limiting amino acid in ruminants from PAA concentrations. mm asvrnv Amino Acid Sam to the Ruminant Nitrogen metabolism in the runimnt has been the subject of sev- eral reviews (Blackburn. 1965: Hungate, 1966; Waldo, 1968: Chalupa, 1968: Conrad and Hibbs, 1968: Smith, 1969; Kay. 1969: McDonald. 1968 and Purser. 1970a,b). The ability of the ruminant to use non-protein ni- trogen is related to its digestive tract which includes large food reservoirs known as the men and reticulum. Forage digestion and non-protein nitrogen utilisation occur in the runon as the result of bacterial and protozoal metabolism. Runon bacteria and protozoa influence the amino acid supply to the animal by degrading dietary protein and by synthesizing microbial proteins (Bryant and Robinson, 1961; Hungate, 1966 and Allison, 1969). Loosli _e_t_. 51. (1949) reported that the essential amino acids (EAA) could be synthesised in the runen of sheep fed only urea as a nitrogen source. Black 33 :1. (1957) and Downes (1961) reported that threonine. valine. methionine. isoleucino, leucine, pherwlalanine. lysine and histidine were essential amino acids for ruminants since they were not synthe- sised in the rumilumt's tissue. The quantity and quality of protein reaching the duodenum of the ruminant depends upon the degradation of dietary protein and the quanti- ty of microbial protein synthesised in the men (Suith, 1969) . Clark _e_‘_t _e_._l. (1966) using sheep cmpared the dietary nitrogen (N) intake to the quantity of N reaching the duodenum. When diets containing 3 4 5.1. 7.3. 16.“ and 2MB gm N/day were fed. 9.0, 12.2, 17.6 and 19.2 gm l/day, respectively reached the duodenum. The quantity of u reaching the duodenum increased on the two lowest ll diets and decreased on the highest N diet in respect to the dietary N level. The amino acid com— position of abomasal contents differs from the dietary amino acid com- position (Clark _e_t_ _a_l.. 1966; Bigwood. 196A! and Little gt 51.. 1968). Clark at _e_._l_.. (1966) and Bigwood (1961+) reported that lysine. threonino and isoleucino formed a larger fraction whereas proline, arginino and leucine formed a smaller fraction of the total amino acids in duodenal digests than they did in the diet. Little _e_t _al. (1968) reported the amino acid composition of abussal digests to be similar in sheep fed soybean meal, gelatin and casein but different in sein-fed sheep. The quantity of undegradod dietary protein which passes to the duodenum was 10 and 60%, respectively, when casein and sein were the dietary proteins fed to sheep (McDonald and Ball, 1957: McDonald. 1954 and Ely _e_t_. _al. . 1967). Solubility determines whether a protein will be degraded by microbes in the rumen. Annison (1956) investigated ruminal degradation of casein, soy, ground-nut protein, aein. bovine serum albumin and wheat gluten and found rapid hydrolysis of the first three proteins. This was substantiated by Hendricks and hrtin (1963) who reported a correlation between protein solubility in a salt solution and rate of degradation in the rumen. Treating protein with heat, tan- nic acid and formalin will decrease the solnbility and increase the quantity of dietary protein passing to the runen (Chalners gt _a_l_;.. 196M Driedger and Hatfield. 1970: Ferguson _e_t_ _a_1_.. 19673 Tagari e_t_ _a_l;., 1962 and mm... _._g_ 5;" 1967). Hangate (1966) reviewed data for protein production in the rumen 5 and estimated production as 15 gm of microbial cells for each 100 gm of substrate fermented by a mixed microbial population. A cell yield of 21.3! was reported (Hungate, 1963) for Ruminococcus Que grown on cellobiose media in continuous culture while, a h6.8$ cell yield was observed for Bacteroidos fllomlus when grown on maltose media (Robson and Somers. 1967). walker and Nader (1968) measured microbial growth rates using radioactive 358 sulfide and founda 1% cell yield for a mind rumon population in 2133. Conrad _e_t_ 2.1. (1967) Published estimates of ruminal methionine synthesis based upon the incorporation of labelled 35: into methionine. Between 31 and 59 mg of «tum. were synthesised per kg of body weight per day by the dairy cow. This corresponds to the synthesis of Lil-2.6 gm of bacterial protein per kg/day. El-Shasly and Hungate (1966) used the. diamincpinelic acid concentration of bacteria to estimate that 69-8” of the total N in the rumen was of microbial origin. The quality of both microbial and undegraded dietary protein which reaches the small intestine will determine the amino acid supply to the rumimnt. Purser (1970b) and waldo (1968) reviewed data regarding quality of rumen microbial proteins. McNaught _e_t gl_. (1951+) and Bergen 23 5;. (1968a) agree in their ostintos of the quality of rumon proto- soal and bacterial p-otoins fed to rats. McNaught _e_t_ £1. (1951+) re- ported the ”true digestibility” (TD), biological value (BV) and not protein utilisation (HPU) as 7“, 81 and 60; respectively, for rumon bacterial protein and 90, 80 and 73; respectively, for rumon protosoal protein. Since the digestibility of protcsoal protein is higher than that of runen bacterial protein the quality of protein passing to the lower gut may increase as increased proportions of protoscal protein pass to the lower digestive tract. Bergen g a}. (1968b) reported no differences in the TD, BV or NH] of microbial proteins taken fru the rumen of sheep fed different rations. The amino acid composition of rumon microbial proteins has been investigated: Purser and Buechler (1966) reported 22 strains of runen bacteria to have the same amino acid composition. The amino acid cm- position of different runen protozoal preparations are similar (Weller, 1957 and Purser and Buochler, 1966). In comparison to runen bacterial protein, rumon protozoal protein contains a larger proportion of lysine, leucine, phexvlalanine and tyrosine (Purser and Buechler. 1966). There- fore the amino acid composition of microbial protein which passes out of the runen is quite constant. The digestibility and release of amino acids frm individual strains of rumen bacteria were studied with a pepsin—pancreatin _i_1_i. _v_i_t_5_9_ digest system (Bergen 33 3;. , 1967). Since digestibility .r the protein in these strains of bacteria varied between #5 and 85%, the supply of amino acids available for absorption by the animal may depend upon the population of runen bacteria. The quantity of microbial protein which passes to the abcmasum is dependent upon the quantities of bacterial and protosoal protein syn- thesised in the runen. The amount of bacterial protein in the rumon of (faunated sheep is only half of that in defaunated sheep (Phi-c.1- 35 5., 1966). Since protozoa use bacteria as a protein source (Coleman, l967a,b and Wallis and Coleman. 1967), the decrease in bacterial counts of faunated sheep may be the result of protosoal feeding or competition for energy substrates in the feed. Bergen at _a_1_. (1968) determined the limiting amino acid of rumen 7 protozoal and bacterial proteins by feeding rats diets which contained these proteins as the sole N source. Histidine and cystine were the limiting amino acids in the rats fed protosoa and bacteria, respective- ly: leucine, arginine and lysine were also in short supply. These con- clusions were based upon determination of the limiting amino acid as described in the PAA score procedure (McIaughlan, 1964). Since the dietary supply of amino acids is not the same as that reaching the abomasum, the amino acid composition of abomasal digests has been measured. The sulfur amino acids were limiting when abomasal amino acid levels were compared to the amino acid requirements of swine (Poley _e_t_ $1., 1963). Schelling gt _a_2|._. (1967) and Duncan _e_t 3.3:. (1953) reported the amino acid composition of rumen digests after sheep were fed a non-protein nitrogen diet. Upon eXpressing the amino acid com- position of rumen ingesta as a ratio of the amino acid composition of whole egg, histidine and methionine were suggested as being in short supply (Schollins 21°. 2.1.- . 1967). The N passing to the small intestine of the ruminant is mainly protein and protein degradation products produced in the abomasum. The l passing to the duodenum is composed of 6 5-75% amino acid nitrogen, 5-10‘S anonia nitrogen (Clark at 93;. . 1966 and Hogan .93.". 11;. , 1969) and 15% nucleic acid nitrogen (Smith at £1. , 1969). Based on differences between the quantities of amino N appearing at the duodenum and iluem, Clark 3; g_1_. (1966) found L10 gnu/day of amino w absorbed in sheep. Amino acid disappearance does not indicate net absorption since there are endogenous secretions of protein in the gut (Kay, 1969). Hogan (1957) reported abomasal N secretion of 1.2 gm/day and small intestinal 1U secretion (8-12 gm/day) was found by Campbell 233 31. (1961) using 131: labelled albumin in sheep. Basset (1%») concludes 8 from rat and dog studies. that endogenous gut secretions my dilute the dietary protein l-h fold and serve as a homeostatic mechanism to prevent an amino acid deficiency after a single meal. However. this will not prevent amino acid deficiencies when feeding an imbalanced diet for a longer period (Rogers and Harper. 1965). Olmstead g}: _a_l_._. (1966) fed b different protein diets to men and found the dietary protein had some influence on the amino acid composition of duodenal digesta. whereas the major supply of amino acids to ruminant tissue is from absorption through the small intestine. other absorption sites have been investigated. Leibholts (1965 and 1969) reported rumon fluid amino acid concentrations to be less than plasma levels. except in the case of lysine. histidine, cystine and alanine which rose above the plasma levels for short periods of time after high protein diets were fed. Cook. Brown and llvis (1965) inserted a catheter into the right ruminal vein of a goat and a steer before adding 15 gm of glycine to the rumon. The ruminal glycine concentration in this study was higher than the normal glycine levels in rumon fluid reported by Wright and Bungate (1968). There is passive amino acid absorption from the rumon. but it is not a major contributor to the free amino acid pool (Kay. 1969). Hogan and Phillipson (1960) reported the disappearance of 0.5-2.0 gm of l/day from the large intestine of sheep. but amino acid absorption from the large intestine has not been established. The N which dis- appeared hem the large intestine was probably absorbed as anonia, transported to the liver. incorporated into urea and the urea excreted in urine or recycled to tho rumon via saliva or by diffusion across the rumen wall (Somers. 1961: Weston and Hogan. 1967 and Houpt and Houpt. 1968). Kay (1969) postulates back-flow of ingesta frm the caecum 9 to the ileum. however a significant contribution to the amino acid pool in this manner is questionable and unsubstantiated. Although protein digestion and amino acid absorption in the small intestine of the ruminant is not known to differ from that in the mono- gastric there are several physiological differences. While .flow through the small intestine of the monogastric subsides as the stomach empties. flow through the small intestine of the ruminant is continuous through- out the day as ingesta continually passes from the rumen (Balch and Campling .1 1965). Therefore there should always be some protein avail- able for digestion in the small intestine of the ruminant. The volume of ingesta which passes into the duodenum of sheep is about 10 liters/day. The major contributors to this large flow are 6-16 liters of salivary and “-6 liters of abomasal secretions (Phillipson. 1964). Honogastrics absorb most of their metabolisable energy from the small intestine as carbohydrate. and ruminants acquire theirs from the rumen in the form of acetic. propionic and butyric acids. These acids are end products of the rumen fermentation and serve along with sull quantities of intes- tinally absorbed carbohydrate to fulfill the energy requirements of the ruminant. The amino acids absorbed from the small intestine may be more efficiently used for productive purposes by the ruminant if meta- bolisable energy substrates are absorbed at the same time (erser 1970a). Dietary protein utilisation in monogastrios depends upon several integrated factors: amino acid composition of the protein. rate of movement through the intestinal tract. intestinal digestion. amino acid absorption and availability of energy sources (mnro. 1961!). The amino acid composition of infusions into the small intestine of man influences amino acid absorption (Orton. 1963 and Abibi _e_t_ 5);. . 1967). The pattern 10 of amino acids released frma protein during enzymatic digestion in the abalasum and small intestine may influence subsequent amino acid absorption. Bergen _e_t_ 2.1;. (1967) reported that arginino was not re- leased from either protosoal or bacterial proteins during a 2 hour pepsin digest _i_n_ v_i__t_r_9_. The consequence of this upon amino acid absorption is unknown. Methods of DetenininLthe Limiting_hmino Acid Determination of the limiting amino acid is an integral part of protein quality evaluation because this identifies the amino acid which limits the utilisation of the other amino acids for protein synthesis. The limiting amino acid is defined as that which, by quantity, is least able to meet the animal requirements. It is the acid that will give the greatest growth response when supplemented into the diet. The limiting amino acids of dietary proteins were first determined by measuring growth response when different amino acids were supplemented into the diet (Mitchell and Salts, 1932). Knowledge about both the dietary amino acid composition and the amino acid requirement of the animal should allow determination of the limiting amino acid by direct comparison (Black and Mitchell, 1911647). The limiting amino acid should give the smallest value when expressing the dietary amino acid levels as a percent of their requirements. The studies of Becora and Hundley (1951) and Sauberlich gt :1. (1953) showed that the limiting amino acid was not always so identified. Carrol 23'. 23:0 (1953) used the amino acids available from digestion in the intestine, as determined from chronic oxide-amino acid ratio in feed and small intestinal ingesta, in conjunction with the amino acid requirements to predict the limiting amino acid. This procedure is lengthy and has not been accepted. 11 Microbiological assays used to access protein quality (Ford, 1962; Miller _e_t_ a_l_.. 1964, 1965b and 1965c) are dependent upon the use of specific micro-organisms which require the presence of one specific amino acid for growth. The amount of that amino acid in the protein is directly proportional to the growth of the micro-organism. This measures the utilisation of an amino acid in a protein by the micro- organism, which my differ from utilization in mammalian systems. These investigators concluded that this method was not sensitive enough to determine the limiting amino acids of dietary proteins. Plasn amino acid concentrations may (Hclaughlan and Morrison, 1968; Richardson gt g._l_.. 19538 Charkey 33; 3.1., 1950 and Guggenheim _e_t_ _a_l_._., 1960) or may not (Denton and Elvehjem, 1951+; Goldberg and Guggenheim, 1962; Hclaughlan, 1963 and Frame, 1968) reflect the dietary amino acid levels. Differences in PAL patterns after feeding protein diets can be attribu- ted te the digestibility of the protein fed (Denton and flvehjem, 195‘! and Goldberg and Guggenheim, 1962). PAL concentrations can reflect the amino acid composition of dietary proteins, but a direct relationship is not always observed. The metabolic state of the animl will influence the PM levels independently of dietary protein. Charkey 9_t_. a_1_. (1953) reported ele- vated levels of lysine and threonine during periods of catabolism and postulated that these amino acids are resistant to deamination. Munro (196“) reviewed data showing increases in PM levels during fasting periods. The presence of readily metabolisable energy, fed to rats (Bergen and Purser, 1968 and Inipfel _e_t_ £1. , 1969), chickens (Hill and Olsen, 1963) and humans (Harris and Harris, 191W: Munro and Thompson, 19538 Swendseid _e_t_ $1., 1967 and Crofford _e_t_ g._l_.., 196“) or infused into 12 the rumen (Purser _e_t _a_1_., 1966) or carotid artery of sheep (Potter at 91., 1968). resulted in decreases in the PM concentrations. The plaslm concentration of the limiting dietary amino acid remained low until the total dietary level of that amino acid was increased above the animals requirement after which the plasma concentration increased linearly with supplementation levels (Zimmerman and Scott, 1965). 31- milar responses were reported for pigs fed a test diet for one week but not for pigs fed the diet for only one day (Mitchell _e_t_ g._1_., 1968b). These data suggest that a period of metabolic adjustment is necessary before PM level will reflect the dietary amino acid composition. Interrelationships between the blood levels of different anino acids have been observed (Kunta and Harper, 1962). Removal of one amino acid from the diet, causing a severe amino acid deficiency, will result in a low plasma level of that amino acid whereas the blood levels of the other acids will increase. The increase in the blood levels of the other amino acids is the result of a lower utilisation rate when protein synthesis is impeded by an amino acid deficiency. Thus, high blood amino acid concentrations do not always indicate that the anilml is absorbing large quantities of amino acids. The relative distribution between PAA levels or changes in levels resulting from the diet may be useful in determining the limiting amino acid from blood. Plasma amino acid concentrations are the measured parameter in several procedures which determine the limiting dietary amino acid. Longenecker and Hausa (1959) used ”plasma amino acid ratios” to predict the limiting amino acid of several dietary proteins. The plasma amino acid ratio procedure predicted the same amino acid to be limiting as had been previously determined in rat growth response trials. 13 These plasma amino acid ratios were calculated according to the follow- ing formula. Plasna amino acid ratio a 5—5-3- Hhere A a PM concentration after consumption of a meal B 8 PM concentration during fa st C I: amino acid requirement of the animal Theoretically the difference between “fasting“ and ”after feed- ing” PM concentrations should reflect the amino acid composition of the dietary protein. When these differences are expressed asa percent of the amino acid requirements, the lowest value should correspond to the limiting amino acid or the amino acid least able to fulfill the requirement. Hill and Olsen (1963) used a similar method for predicting the limiting amino acid of protein fed to chickens. PM concentrations after feeding a non-protein-calorically adequate diet were used in place of the fasting PM concentrations. This procedure predicted the same amino acids as limiting in sein, gelatin and casein diets as did growth trials. The ”plasma amino acid score" procedure has identified the limiting amino acid of many proteins (Hclaughlan. 1964: Hclaughlan. 19673 and Rao e_t_ 21., 1968). Plasma amino acid scores are defined as: Plasma amino acid score a A x 100 B Where A 8 PM concentration of fed animal B = PAA concentration of fasted animal kith and Scott (1965) caspared PM concentrations frma chickens fed a reference crystalline amino acid diet, shown to support optimal growth, to PM concentrations from chicks fed test protein diets. The limiting amino acids in sesame meal and soybean meal were identified by: 11+ _A_«_-_§ When A I PAA concentratioi for animals fed test diet B 8 PAA concentration for animals fed crystalline amino acid diet While three of these procedures have never been extended beyond the developental stage, the plasma amino acid score procedure (McLaughlan, 1961+) has been easy and extensively used in determination of the limiting amino acid of proteins fed to rats. While its use has not been extended to other aninls, it has been very accurate in identifying the limiting amino acid as determined in growth trials. Elana Amino Acids in Ruminents The FAA methods (Longenecker and Reuse, 1959: Hill and Olsen, 1963; McLaughlan, 1964; and Zinerman and Scott. 1965) used to determine the limiting dietary amino acids in monogastrics cannot be used in rumin- ants. The methods of Longenecker and Hause (1959) and Hill and Olsen (1963) require both knowledge of the amino acid requirements of the aninl and, either fasting PAA concentrations or PAA concentrations after a non-protein meal. The PAA score method (McLaughlan, 1964) re- quires fasting PAA concentrations whereas the method of anith and Scott (1965) requires a reference diet composed of crystalline amino acids (shown to promote optiul growth of the chick). These data are not available for the ruminant. Purser _e_t_ a}, (1966) suggested using a modification of the Hill and Olsen (1963) method to determine the limiting amino acid in ru- minants. Plasma amino acid concentrations increased after feedirg a low calorie diet whereas a high caloric diet decreased the PAA levels (Parser _e_t_ 91.. 1966). Decreases in PAA levels resulted from intra- arterial infusion of glucose and propionate (Potter _e_t_ 11.. 1968). 15 Purser e_t_ _e_l. (1966) expressed the PAA levels'after a starch-glucose in- fusion as a percent of the pro-infusion level, and Potter _e_t_. e_l. (1968) expressed the post-glucose PAA levels as a percent of the pro-glucose levels. These infusions of glucose or starch and glucose appeared to result in protein synthesis since the decreases in the essential PAA levels correlated to the essential amino acid cmposition of striated muscle of lamb. The amino acid which decreased the most during protein synthesis (relative to tho original levels) should be the limiting amino acid. The limiting amino acid so determined was lysine for defaunated sheep whereas . no single amino acid was limiting in faunated sheep . (Purser at .51.. 1966) and isoleucino was limiting in glucose infused sheep (Potter _e_t_ $1.. 1968). The effect of a metabolic energy shortage upon the PAA levels that .occur in the fasting state, was reported by Leibholts and Cook (1967) . ' in sheep and by Brown gt _a_2_|._. (1965) in cattle. Three weeks of starvae- tion decreased the plasma levels of threonine, glutamic acid, alanine. valine. isoleucine and leucine whereas lysine levels increased (Leibholts and Cook. 1967). The plasma levels of phenylalanine, tyrosine, citrulline. ornithine. lysine. valine. threonine. leucine and iso- leucine increased gradually over an 88 hour fast (Brown e_t_ 51.. 1965). The differences between these studies may be due to length of fast as the source of energy to the animal may change with length of fast. The amino acid composition of abomasal contents, which indicates the amino acid supply to the small intestine of the ruminant was reflec- ted in the PAA concentration pattern of these sheep (Paley and Trenkle. 1963 and little e_t_ E" 1968). Hogan gt: e_l_. (1968) reported that plasma essential amino acid levels increased as increasing quantities of casein were infused into the duodenum. 16 Increases in wool growth (Rois and Schinckel, 1964; Rois, 19693 and Colebrook and Rois, 1969) and nitrogen retention (Egan, 1965 and Schelling at $1., 1968) has resulted fraa the abomasal supplementation of protein and/or amino acids to sheep. Results from these studies sug- gest that either protein or an amino acid is limiting productive per- formnce of the unsupplemented animals. Study of changes in the plasma amino acid levels of ruminants abomasally infused with protein and/or amino acids may be a useful guide in future limiting amino acid determinations. Schelling (1970) supplemented methionine or casein into the abomasum of growing lambs fed a soybean meal diet (methionine is limiting in rats fed soybean protein) and measured N retention and PAA concentrations. Lambs fed a 11% crude protein diet and abomasally supplemented with 0, 1.0, 2.0 (and 3.0 gm of dL-methionine or 30 gm of casein/day had positive N balances of 3.74, 3.94, 4.12, 4.25 and 5.12 gm/day respectively, where- as lambs fed a 14% crude protein diet and receiving either 0, 1.0 and 2.0 gm of methionine had positive N balances of only 1.46, 2.07. and 3.11 gm/day respectively. These results suggest that methionine was the limiting amino acid in sheep fed the 14% protein diet and protein was limiting the 11% group. Plasma methionine levels of 3.7, 6.9, 11.9 and 39.3 micrograms/m1 were reported for lambs abomasally infused with 0.0, 1.0, 2.0 and 4.0 gm of dL-methionine/day, respectively (Schelling, 1970). The increase in plasma methionine levels, which resulted from the supplementation of 4.0 gm of methionine, suggest that the methionine supply had exceeded the requirement. Thus, methionine was the limiting amino acid in unsuppluented animals. Ely _e_t_ $1. (1969) reported an increase in the plasma lysine and a decrease in the other PAA levels when lysine was intravenously infused into sheep fed a coin diet 17 (lysine is limiting in sein fed rats). Thus lysine was suggested as limiting in the lysine unsupplemented sheep. This approach, of titratation with the eXpected limiting amino acid until there is an increase in the plasma. level of the supplemented amino (acid may prove useful in determining the limiting amino acid in mminants. In this system an increase in the plasnm concentration indicates the amino acid is no longer limiting. orator and Fraser (1969) reported N balance data for lambs bottle- fed liquid protein diets. When fed through a nipple, the diet will pass directly to the abomasum and avoid ruminal modification (firskov and Fraser, 1969). The use of liquid protein diets my aid in the develop- ment of a method for determining the limiting amino acid in ruminants as this would eliminate the modifications of the dietary protein in the rumen. The present research was undertaken to investigate PAA concentra- tions afi changes in PM levels which result from glucose infusion as these may be useful in determining the limiting amino acid of ruminants. Theoretically, PAA concentrations reflect the ”tissue pool” of free amino acids (Rogers and Harper, 1968) which, in turn, are influenced by the supply from absorption and by utilization for protein synthesis (Munro, 1968). The rate of incorporation of amino acids into protein is influenced by energy availability for protein synthesis (Munro _e_t_ 51.. 1962). Assuming that the PAA concentrations reflect the amino acid supply in ruminants, and that glucose infusion will stimlate protein synthesis should show the largest relative decrease in the plasma immed- iately after glucose is infused. This relative decrease has been expres- sed as ”plasma amino acid'index" (PAAI), which are calculated 18 according to the following formula. 8 PAA concentration post-Egcose PAA concentration pro-glucose PAAI x 100 The limiting amino acid for the ruminant should be indicated by the lowest index value. METHODS AND MATERIALS Blood Samples for Amino Acid Anemia Blood samples were collected in heparinised syringes and the plasma separated by centrifugation in a refrigerated centrifuge at 10,000 x g for 10 minutes. To each ml of plasma, .1 micro mole of norleucine was added as an internal standard. After mixing, .1 ml of 50% (w/v) sulfosalycylicacid, per m1 of plasn, was. added to. precipitate plasma proteins. After centrifugation at 25,000 x g, the protein-free filtrate was frozen and stored until analysed. Plasma amino acid concentrations were determined with a dual column TSH-l Technicon Auto Analyser using a procedure developed in this laboratory and reported by Hakdani gt :1. (1970). Emeriment (he This experiment was designed to substantiate the report by Potter _et a}. (1968) that PAA levels of sheep decreased following an intra-arterial glucose infusion. Two groups of three, growing wether lambs were selected on the basis of equal body weight and . surgically prepared with exteriorised carotid arterial loops, accor- ding to the method of Bone _e_t_t _a_l_.. (1962). The lambs in one group (replicate 1) each weighed 14.9 kg whereas the lambs in the other group each weighed 12.3 kg (replicate 2). Within each group, the lambs were randmly assigned to a 3 _x 3 latim square; treatments were saline, low glucose and high glucose. Replicates l and 2 were 19 20 repeated and these are referred to as “replicates 3 and 4", respectively. There were 15 days between replicates and 5 days between collection days (periods) within replicates. The experimental design is shown in Table l. lambs were housed indoors, singly, in 4 ft x 6 ft, metal stalls and had free access to water. They were fed daily (8 a.m.) a portion of diet 1 (composition shown in Table 2) equal to 7% of the metabolic body weight (Bins) (Kleiber, 1961). (h treatment days, feeding was delayed until after the second blood sample was collected. _At 8 a.m. on treatment days, 10 m1 of blood were withdrawn from the carotid artery into a heparinised syringe and then treaments (saline or glucose) were infused into the jugular vein via catheter. A second blood sample was collected one hour after the infusion. The 8 a.m. blood sample is referred to as the ”T " sample and the pest infusion 0 sample as ”T1". The treatment infusion mixtures were; .9% RaCl (W/V), low glucose (25% W/V) and high glucose (50% W/V): these glucose infusions provided glucose at .05% and .1% of the metabolic body weight (BW'75), respectively. Infusion volumes were 29.8 ml and 24.6 ml for the heavy and light groups of sheep, respectively. Experiment Two I The purpose of this experiment was to determine if the PAAI method (Potter _e_t e_1_. . 1968) would identify the limiting amino acid of dietary proteins fed to rats. Four diets (Table 3) were prepared with egg albumin, casein, soya or sein as the protein sources. Forty-eight, 160 gm white male rats were randomly assigned to four diets. We rats were housed in each cage. Rats were fed between 8 a.m. and 5 p.m. for a period 'of 2 weeks. 21 Table 1 Experimental Design for Ekperiment One w'S—heep no. fieep no, Period Replicate 2 it 6 Replicate 8 i ll 1 1 x‘ I z 2 x I 2 2b 1 Z X Y 2 I Z X 3 l I Z X 2 Z X Y 1 3° 1: I z 1+ x Y z 2 3 Z X Y 1+ Y Z X .2 J Y Z X ‘4' Z X Y ‘Indicates the treatment: 1”Five days betwaen periods c’F‘ii‘f‘lzeen days between replicates = saline: I = low glucose = high glucose 22 Table 2 Rationg, Experiment One Ingredient Percent Corn cobsb t"5.0 Alfalfa meal, 17$ CF0 35.0 Whole rolled oats 12.6 Cane molasses 5.0 Urea, 262$ 09 0.1+ Dicalcium.phosphate 0.94 Trace mineral salt, high Zn 0.94 Sodium.sulfhte, anhydrous 0.09 Vitamin A premix, 10,000 IU/gm 0.006 Vitamin D premix, 9,900 IU/En 0.002; tion contained 1.90% nitrogen Findersons' No. # Fines, The.Andersons, Haumee, Ohio. Remainder of cob after hard cylinder has been removed for production of industrial.abrasives. Consists of bracts and pith (soft parenchyna without vascular bun- dles). Cell wall constituents, 81.2%; acid detergent fiber, 37.5% lignin. 6.5%. 0Crude protein 23 RN.» a 5 2.3.3 .3 85-...»er £8 .5: 5926 ten Heed-283 Hts—co .823 NE fiapuoflflsom ago 5 IE on 3a fin omen 38 .5: £216 .63 Hans-n88 stones spa 858.8 on... :28. £38... can 93 add 13 With .25 £888 .H. .H. .a. .H. and-3.9 : s a a nuaem n n n n duo Eco in 3m 3m in emoaeheo an on on an n23. 8.8 mm e55 8 atom cm .53; and: .588 mm I; «5557 new dawn {mm flomuo snug—ma new one; eon—6e 530mm 2.9 23483 .335 n Ana. 24 Following a 16‘ hour fast on the 15th day, one rat in each cage was given glucose (50$ HIV) and the other (.91 Na01 W/V) by stmach tube. Infusion volumes were equal to .002 ml/gm of the metabolic body weight (W75). One hour after the infusion, each rat was anesthetized with ether and blood was collected by heart puncture with a heparinised syringe. Experiment Three This experiment was designed to evaluate limiting amino acid determination by expressing PAAI from rats fed different proteins as a percent of PAAI of rats fed a high quality protein. Eight different protein diets (Table 1+) were prepared using whole proteins and crystal- line amino acids. Eighty, 150 gm rats were assigned to treatments and housed as described for experiment two. EXperiment 3 differed from experiment 2 in the following ways: (a.) 10 rats were used per dietary treatment instead of 12; (b.) rats were fed for only one week instead of two; (c.) rats were fasted 8 hours instead of 16 hours: and (d.) 4 ml of saline or 50% glucose were administered regardless of weight. firinent Four This experiment was designed to study PM concentrations and glucose induced changes in the PM levels of sheep duodenally infused with proteins of "known" _ amino acid composition. Eight sheep, weigh- ing between 35 and #5 kg, were surgically prepared with both rumen (Jarrett, 1948) and rec-entrant duodenal. cannulae (modified from procedure described by Harkowits _e_t_ 93.. 1961:). A 15 cm long skin incision was made 3 cm below, and parallel to, the abdminal ends of the 25 38 83s £356 . :5 ”.03383 3.850 683 .5: 83833.5 a3“? .33 .303 .35 4. tan mu £an 3 v.3 2 and :3: H233. .a 553.0 :8 23333.! 033m .04 39¢ 3539.. :5 3,2. Sam Econ 36 33¢ 5&5 . .oaH Haas-.803 3.525 sec 353% 8:. £38 8395 nuns e5 finned no. .355 .133 35.6.38 .3 v.8 3 ads «€333.33. ounce no and: a can...“ .Lm eehmH pneaduenwm .eeeda.aspneadheaxm 3.0 3.0 Bandeau N10 «.10 810 3.0 efiuoénq . «0.0 «5.0 90.0 3.0 3335an was $30 on... 86 afichAnfifii mN.0 mN.0 00.0 endahquq 3.0 3.0 3.0 3.0 gauged—mug a .n a H H H H H sung-pg a a a a a n a 0 0:35”! fluem n n n n n n n m 36 5.8 mm mm mm mm mm nu mm mm eeoaoueo mm mm mm mm 5 m: .3 n: has». 58 3 3 3 3 .8 non dz: 53%... as one mam Heel_=epsam :uoo «N as «.8 58.93 83 .388 mo «N Ru *3 fiopoa mm. .36. u a u u a u u a 38383 1+0... 1+0m 11.5% mom :8 So «4+8» 30.- ends.- 33 cause odes cud-e Heew Hoes 5826 30s end!- we Heel Heel Heel seephom s33» Eco mme 325 odes; naenhom seenhom seenhom n a L To 3 H1 i... 26 Inst three stetionery ribs on the right side of the sheep. Access to the ebcnesel-duodeml junction wes gained by either cutting through the nuscles end peritoneun or by spreeding the muscles end then cutting the peritoneun. The small blood vessels, loceted eround the duodenum end 5-? cn beyond the eboneeel-duodenel junction, were ligeted with 00 chronic gut. Two Kocher clenps were pleced edjecent to eech other end the smell intestine sectioned between these clenps. The ends of the trensected smell intestine were closed by ”oversewing" eech clenp end then edding e second suture row for reinfomenent. The enterior cennule was located in the duodenum nidwey between the ebolesel-duodenel junction end the closed end of the duodenum. nu posterior cennule wes loceted in the duodenum 12 cm beyond the point where the cannon bile duct enters the duodenum. The posterior cennule wes inserted efter making e 6 on long incision through the skin, muscles end peritoneum. Both cennules were pleced in the nell intestine through 2 cu long incisions. Chronic gut purse-string sutures were aligned eround the pereneter of the incision, end then tightened to hold the cennule. A second set of purse-string sutures were used for reinforcement. The heed of eech cennule wes exteriorized through e steb wound 1 inch laterel to the incision. The incisions were closed end the cennulns connected. Cennulee were nede from .0M inch thick silestic sheets, rein- forced with decron nesh, end silestic nedicel edhesive type A (both ebteined Ira Dow Corning Corp, Hidlnnd, luchigen). The cennule nold wee nude from two pieces of copper tubing 3/8" 0.D. end 3}" long. The end of one of the pieces wee cut so thet it would fit 1/3 of the wey eround the other piece when they were et e “5° engle 27 and flat on the table top. The two pieces were soddered together so that the end of. the cut piece was attached to the mid point of the other piece. The nold assumedits final shape when the free end of the neck piece was bent downward toward the base piece, a distance of one inch. After the sheep were on full feed for one week, the connection between the two duodenal cannulas was removed and a protein-mineral infusion was pumped into the posterior cannula whereas ingesta coming from the abcnasun passed to the floor through the anterior cannula. The compositions of the protein-mineral infusions are shown in Table 5. The infusion mixtures were prepared by bell—lining the protein g. (not necessary for egg) , homogenizing the protein-nineral-vitanin—fat mixture. adjusting the pH to below 3.0 with 6N 301. adding 1 gal of pepsin and diluting to 6 liters with tap water. Each infusion was punped continuously over a seven day period so that 6 liters were infused each day. The number of sheep per protein infusion is shown in Table 5. Glucose treatment and blood collections were on days 1+ and 6 of each period. Twolve ml of blood were withdrawn with a heparinised syringe from the jugular vein at 8 am. This was followed inediately by the infusion of 30 :11 of 35$ (VI/V) glucose into the jugular vein via a catheter. helm ml of blood were collected one hour after the glucose infusion. These blood samples were designe- ted as ”To” and "T1" for pre- and post-glucose infusion, respectively. Sheep were offered 800 gm of either ration 1 (Table 2) or a ration of cracked cornand rolled oats. If they consuned loss than 200 gm of their diet, 400 g- of the diet was infizsed into the rumen through the runen cannula. These eninels were fed at 8 a.m.. except on 28 0 sand. 5 was»: 3 83283.3 Au? dd 2A 38238 e5 3.0 58 no chafing 38 .afla 5356 55 102288 H228 .803 $3 83333.86 £59» .a .5388 .8 mfiufiodéfi soda .mJ 8h eofifino .mo *3 :5 13a :33» Econ .HHH omeodnu ..onH whom Hegvcoo any“ vocauvno evanescence adopohm memo one .33 c325 :oaH Hzgnooa Hanson 39¢ 353% 982 5.551 mom 28 fiends “583: .Boflmfihm 68a 53:55 H5 353% mes £2? 0.338 23:89 _heu hem seamsnnfi no em ed beveda one eenmup dado eoanup psoavonnsn ad peas ecoflpewpounndi a H W a a m a n scamsmfiwmoofi no Sega n6 n6 n6 n6 n6 m6 m6 n6 “HE its? 3.5. 3 S S S S S S S oumm . «8»: on on on on on on on on o m: «2 d” 3 fl 3 S 3 fl 3 98 ea ea ea aa :a ea 3H 2H Houz mm mm NN mm mm mm mm mm 8a m m w w w m w w Avg N m N m m m m N masses: {m ofiqofififide mm «Hues nevaam anon on 5:51 mmm a 2. u .ssaé on e538 CAN 03a _ com ammo eHonz am: aw m: «m was +Hom :8 mom 98 um: pcofieammmm an mwe MN mme cascade ecflzodnvos.+ Hues honoaonm adomuo mMm MHbgz odes: mwm hoaoaoAMIi, seesaw choc odes: t! l; .832 ham eaoafié £883: consume." atone: 530& m .Sa. 29 Table 6 Trace Minera1.Hixture, Emperinent Four 1952022212 xc1 [I Pusan 0 2H20 CuSQg Co003 Hn304,' 320 ZnSOh - 7H20 H8C03 NIHCO3 CaHPOh ' ZHZO Ca003 Cerelose Percent 10.0 ,0.002 0.7 0.1 0.1 0.1 0.‘ 2.0 25.0 36.0 12.5 12.928 100.000 30 sample days when feeding was delayed one hour until after both blood sasplas were collected. Eperiment Five This experinent was disigned to determine if blood collected from the jugular vein differed in PM levels from blood collected from the carotid artery of sheep. Three. two-year old wethers. which had been surgically prepared with exteriorised carotid arterial loops (Bone _e_t _e_l. . 1962), were used in a 3 x 3 latin square experiment. The sheep were fed 800 gm of maintenance ration (composition shown in Table 7) daily at 8 a.m. , for 2 months proceeding and through out the experiment. The second and third collection days followed the first by 7 and 11} days. respectively. (h collection days feeding was delayed until after all blood samples were collected. At 8 am. on collection days. 10 m1 of blood was withdrawn sinul- taneously from the carotid artery and jugular vein. Thirty ml of either saline (.9$ RaCl. H/V). glucose (30% W/V) or acetate (30% W/V) solutions were then infused via a catheter into the jugular vein. The pro-infusion and post-infusion blood samples were disignated as ”To” and ”T1”, res- pectinly. Statistical Analysis All data reported in this dissertation were analysed on an IBM 3600 computer at the Michigan State University Computer Laboratory. Least squares analysis of variance (Harvey, 1960) were used in experi- ments 1, lb and 5 while analysis of variance procedures were used in experiments 2 and 3. Duncan's new multiple range test was used to determine mean differences (Steel and Torrie. 1960). An example of 31 the analysis of variance and Duncan's new multiple range test pro- cedures is shown in Appendix I. 32 Table 7 Ration" . Experiment Five Ingredient Percent Ground corn 32.0 Ground hayb 30.0 Cane molasses 5.0 Ground corn cobsc 30.0 Dicalcium phosphate 0.91! Trace mineral salt, high Zn 0.9“ Sodium sulfate, anhydrous 0.09 Vitamin A premix 10,000 IU/gm ' 0.006 Vitamin ILgremix 9,000 191m 0.002 Contained 1.36% nitrogen bMixture of grass and legume hay cinderson's N0. 1+ Fines. The Anderson. mumee, Ohio. Minder of cob after hard cylinder has been removed for production of industrial abrasives. Consists of bracts and pith (soft parenclmna without vascular bun- dles). Cell wall constituents, 81.2%: acid detergent fiber. 37.5% lignin. 6.5%. RESULTS Emu-ant me Mean pro-treataent (To) and one hour post~treatment (T1) PM concentrations are shown in Table 8. There were no differences among the individual PM levels of the saline, low glucose and high glucose groups at T0. The low glucose infusion significantly (P < .05) de- creased the T1 plasma levels of all essential amino acids (N) except threonine. methionine and histidine whereas the high glucose infusion significantly (P < .05) depressed all EM except threonine. Total TO plasn EAA levels were 10.77, 1%!“ ad 15.12 mg/100 ml, whereas total T1 concentrations were 13.01, 10.70 and 9.64 sag/100 ml respective- ly for the saline, low glucose and high glucose treated sheep. These PAA concentration are similar to those reported in sheep fasted for 2‘} hours (Potter _e_t_. 11.. 1968). Potter L“. 3.3:. (1968) expressed T1 PM concentrations as a percent of To PAA concentrations, and referred to these as plasaa amino acid m... (PAAI). PAAI, which express the change in m due to treat- ment are shown for this experiment in Table 9. The average PAAI fer the EM were 88, 7*} and 6“ whereas the average non-essential PAAI were 85, 85 and 81 for the saline, low glucose and high glucose treahents, respectively. Thus the decreases in the plasma EM levels were 12, 26 ad 36 percent for the saline, low glucose and high glu- cose. respectively. This decrease after saline infusion nust 33 Mean Plasma Amino Acid Concentrations‘l by Treatment and 31.. Table 8 Sample Time for Experiment One 1 sample time Pro-treatment (To? Post-treatment (Ti) Amino 130w _High Iow High thm _SEL Salmfslwm £1114 _ Essential amino aci s m- 1.86 1.69 1.77 116 1.62 d 1.32b 1.33 3.22 v.1 3.02 3.01 3.12 1.22 2.71“ 2.29 2.19: 2.25"c not .35 .39 .37 2.06 .29‘ .29“ .22 1.09- 11. 1.33 1.30 1.39 $.13 1.16‘ll .86b .70" 1.12“ Lou 1.73 1.72 1.86 1.21 1. 51ll 1.14: .90: 1.17" Tyr 1.04 1.03 1.05 1.06 .95;l .75b .67 1.12" ”1. e70 07” e72 L05 062 .561) .1490 i06" Lys 1.90 1.82 1.95 1328 1.66: 1.43‘ 1.08: 1.15" 111." .78 .78 .75 1.07 .66‘ .63b .59b 1.00M Lrg 2.06 “1.36 2.1% 1.32 1.84 1.0 1.3 1225* Ten" 15.77 1 . 15.12 13.01 10.70 9. Ch‘nge - - - ”le?6 '30?“ "Seas Non-essential amino acids + ‘SP e54 e82 e56 ire—“5 .% a“ e38 ‘e08 Ser 1.55 1.177 1.6+ 3320 1.2“ 1.25 1.30 $.21} Asn .113 $3 .54 $.12 .27 .27 .41 11.07 Glu 2.07 2.47 2.61 1.33 2.13 2.15 1.86 5.30 Pro 1.56 1.33 1.07 1.31 1.15 1.32 .89 1.42 Gly 7.50 7.78 7.02 i386 6.82 6.60 6.01» 5:.51 11. 2.20 2.24 2.00 35.27 1.90 1.78 1.76 5:.26 Cys .22 .24 .23 1.05 '2}. .21.:2; 1:22 Orn 1. O 1.12 . Z 4.7.58 .9 1.12 . t. TNEAA‘ 17.81 1'71'9’0‘ 17.18 15.16 15.23 13.90 Change - - - -2.65 -2.67 ~3.28 wg 32558 32.34 32530 28.11 25.93 3.9+ ‘All values are expressed as mg/100 ml b'I‘he histidine values are from replicates 3 and it only “Differences between the means were significant (P < .05) MDifferences between the means were significant (P < .01) d grouping (P (.05) “Total essential amino acids fTotal non-essential aaino acids gTotal amino acids Values with similar superscript form a statistically homeogenous 35 Table 9 Mean Plasma Amino Acid Indicesa’ by Treatment Experiment One Treatment Amino Low , High acid Saline glucose g_glucose Thr 8? 78 _' 75 Val 90 76 70 Net 8 3" 70b 59 Ile 87 66 53b Leu 87 66b 51 Tyr 91 73 64 Phe 89 76 68 £78 87 79 55 His 8 5 81 29 Arg 8 3 Ave EAAIc 8% $73 6% Asp 85 56 68 Ser 80 85 79 Asn 63 63 76 Glu 86 87 71 Pro 74 99 61 GI? 91 85 87 Ala 88 79 86 91$ 95 32 78 0m 72 l 101 Ave NEAAId “8'5. ’85. Tl Ave lAAI° 86 80 73 _ TfPlasna amino acid concentration To Plasma amino acid concentration bLowest EAA index for each treatment °Average essential amino acid index dAverage non-essential amino acid index °Average total amino acid index "Index x 100 36 represent the normal change since the volume of infusion (30 m1) would account for less than a 2 percent dilution in the blood. In comparison to the average decrease in plasma non-essential amino acids, the average plasma EAA were decreased more by glucose. These results confirm the results of Potter _el _a__l_._. (1968) who suggested that glucose caused decreases in the plasnm levels of all amino acids, but that synthesis of non-essential amino acids accounted for the smaller depression in plasma non-essential amino acid levels. The amino acids predicted as limiting (lowest PAAI) were isoleu- cine and leucine for the low glucose group and leucine for the high glucose group. In theory, these amino acids should be the limiting amino acids of the protein in the small intestine of the ruminant. These results agree with previous PAAI predictions which show isoleu- cine and leucine as the limiting amino acid after glucose infusion in sheep (Potter 33 91., 1968). The actual change in the concentration of the plasma EAA from T to T were compared to the EAA composition of striated lamb muscle 0 1 (Block and Weiss, 1956) by linear regression (line of best fit). The resulting correlation coefficients (r) were; .73 (P ( .05). .67 (P < .05) and .82 (P (.01) for the saline. low glucose and high glucose treat- ments, respectively. The correlation coefficients obtained for the low and high glucose treatments may have been influenced by physiological changes in the animalattributable to factors other than glucose. Subtracting the TO-Tl saline (control) concentration differences from the To-Tl glucose concentration differences should give concen- tration changes due to just glucose. Linear correlation analysis between the saline corrected glucose differences and the EAA composi- tion of lamb showed coefficients (r) of .5“ (N.S.) and .80 (P < .01) 37 Table 10 d Differences Between the Pro-Treatment (To) and Post-Treatment (T1) PAA Concentrations Treatment group _— Amino X Law High Corrected differences. acid Saline glucose glucose Y-X Z-X Thr .25 .37 .116 .12 .21 v.1 .31Elf .72‘‘13 .93b .91 .62 Met .06 .10 .15 .00 .09 11. .17‘ .04“ .65b .27 .48 Lou .22‘ .58“b .92b .36 .70 T7!- .09‘ .28“ .38b .19 .29 Phe .08‘ .18“1D .23b .10 .15 Lys .20‘ .39"b .87b .15 .63 His .12 .15 .16 .03 .09 Arg .22‘ .53"b .75b .31 .53 r3 .73 .67 .82 .55 .80 P < .05 P < .05 P < .01 N.S. P < .01 dValues are expressed as mg/100 ml “Corrected differences were obtained by subtracting the differences for the saline treatment (column X) from the glucose groups (columns Y and Z) fValues with similar superscript form a statistically homeogenous ECorrelation between plasma essential amino acid depression pattern and EAA composition of striated lamb muscle. 38 for the low and high glucose groups, respectively. The results of the high glucose treatment confirms observations by Munro and Thompson (1953). Crofford _e_t_ 11. (1961+), Swendseid e_t_ 51. (1967) and Potter _e_t_ 31. (1968) who reported that glucose ingestion or infusion caused a de- crease in plasma EAA levels which was similar in composition to the m composition of striated muscle. This relationship between changes in plasma EAA levels and the EAA composition of muscle suggests that pro- tein synthesis occurred after the high glucose infusion. Despite the suggestion that glucose caused protein synthesis, the accuracy of the PAAI method in predicting the limiting amino acid was questioned for the following reasons. First, PAAI calculated from data reported by Munro and Thompson (1953) and Crofford _e_t g.__l_. (1961+) showed isoleucino had the lowest index when subjects were fed proteins not limiting in isoleucino. Second, methionine is suspected as limiting in sheep since sheep have a high sulfur amino acid requirement for wool growth and since the supply of methionine in bacterial protein is low. Experiment Two This experiment was designed to test the accuracy of the PAAI method (Potter at 11. 1968) in predicting the limiting amino acid in dietary proteins fed to rats. Mean PAA concentrations for 16 hour fasted rats are shown for each dietary group in Table 11. Except for aspartic acid, the plasma levels of all amino acids differed signifi- cantly (P < .05) with dietary protein sources. The relationship be- tween dietary and plasma amino acid levels was examined by determining correlation coefficients. No significant correlations were found when the plans EAA concentration pattern was correlated to the EAA 39 Table 11 Mean(1 Plasma Amino Acid Concentrations of Rats Fed Different Protein Diets, Experiment Two fists “— Amino Egg " Significance acid albumin Casein: Soy Zein SEM of F value c Essential amino acids _____ m- um“ 11.27" 2.86: 11.15: 3.20 P < .001f v.1 1.21: 1.18: .87a .77.) 1.06 P < .001 Hot .21 .20ll .16b .32b 1.03 P < .001 Ile .55: .55“ .qu .383 1:.03 P < .001 Leu .98 .93“Lb ~71. 1.17b $.07 P < .001 Tyr .45: '59. .45b .51c 1.09 P < .05 Ph. .148“ e 3 e37‘ e59b ie03 P < .001 Lys 4.36; 4.84.!) 4.th 3.03,3 1.00 P <.05 His °"°. '53. .60b .886 1.09 P < .001 Arg 1.08 1.0 1.28 1.58 1.08 P < .001 Tan“ '11' .16 13.61! 12.13 13.38 Non-essential amino acids 28p e 87‘ e 6981) e 71b 0 73¢ to 06 no so er 3°20. 3°67ab 4.02b 5'97. 1.19 P < .001 Glu 3.163 2e79ab 2.34“ 3.06b $.15 P < .005 Pro 1. 513 1.62]D 1.36b 1°91. t.10 P < .005 Gly 4.03.L 3.21 b 3.13b 3.83“, 1.19 P <.005 11. 4.18 3.75: 3.11b 3'72“. 1.21 P < .01 Cys .37: J9. .28‘l . b 1.02 P <.05 Orn h .178 .27 .4] .76 1.04 P <.001 THE? 16.93 15.90 17+.71 19.59 m 28. 09 30.51 26.84 2 .97_ dMeans of 12 rats and are expressed as mg/100 ml_ °Values with similar superscript form a statistically homeogenous grouping (P <.05) 1'Determined by analysis of variance 8Total essential amino acids hTotal non-essential amino acids 1Total amino acids 1+0 composition (distribution pattern) of the dietary protein. While there was no significant correlations between the blood and dietary levels of BAA, the limiting amino acid of each dietary protein was reflected \ by a low plasma level of the limiting amino acid. The plasma threonine level.in.rats fed egg albumin was significantly (P <.05) lower than the plassm threonine levels in rats fed the other diets. This relationship was also true for lysine in sein-fed rats, for methionine in soy-fed rats and for arginine in casein-fed rats, however the lysine and methio- nine levels were not significantly lower then their levels in rats fed the other diets. Methionine and arginine have been suggested as the limiting amino acids in casein; the plasma methionine level, in the casein-fed rats was lower than its level in egg albumin- and coin-fed rats. Thus to a limited extent PAA levels alone reflected deficiencies in dietary amino acid intake. However, this relationship is not such that the limiting amino acid can be predicted on this basis alone. Mean PAA concentrations, from rats fed all four diets, after either glucose or saline infusions are shown in Table 12. Glucose infusion resulted in a significant (P <.05) decrease in the plasma levels of valine, isoleucino, leucine, phemlalanine, serine and glycine and increases in plasma alanine and cystine. Mean PAA concentrations for the saline- and glucose-infused rats on each dietary protein are shown in Appendix II. PAAI for each dietary protein treatment were calculated by ex- pressing the PAA concentrations from glucose treated rats as a percent of the PAA concentrations from saline treated rats (Table 13). The amino acids predicted as limiting (lowest index) by the PAAI were isoleucine in egg albumin, leucine in casein, methionine in soy and 1&1 Table 12 Hean‘ Concentrations of PAA According to Saline or Glucose Treatment and Overall PAAI, Experiment Two Treatment Amino Significance of c acid Salji_.n_e _Glucose SEM F value PAAI Essential amino acids m- 3.31 3.05 3.10 n.s.b 92 Val 1.07 .90 35.00 P < .05 88 Hot . 22 . 22 i. 02 N . S . 100 Ile .52 .43 $.02 P < .005 83 Lou 1.10 .79 1.05 P < .001 72 T3? 052 0""9 17.03 Nos. 94 Phe .53 .010 i.02 P < .005 83 Lys 1+. 03 l}. 30 i'. 28 N .S. 107 His .59 .61 1302 11.8. 103 mm“ ""'813. o ”—5612. Ave 12111 '96 Non-essential amino acids Asp .81 .69 1.00 N.S. 85 381' “.56 3e89 $.13 P < .001 85 Glu 2.99 2.69 t.ll 11.8. 90 Pro 1.66 1.50 1.07 N.S. 93 Gly 3.92 3.18 $.14 P < .001 81 Ala 3.20 0.19 $.15 P < .001 131 Cys .31 .38 $.01 P< .005 123 an 02 e 1303 NeSe 102 “E?" 17.99 17.11 Ave mu ‘93 TAA 31.07 29.67 Ave TAAI 26 ‘Hean of 29 rats and are expressed in mg/100 ml bDetermined by analysis of variance °PAAI are glucose group values divided by saline group values x 100 d'l‘otal essential amino acids °Tota1 non-essential amino acids otal amino acids f T #2 Table 13 PAAI‘ for Treatnents in EXperinent ‘l‘wo ' Keir-l treatment Amino Egg acid albumin Casein _fiSgL __ Zgig m 101 97 98 83 Val 85 81 ' 99 106 not 115‘c 29 61" 106 Ile 72b 77 98 88 Leu 76 69b 88 77b Tyr 86 105 109 87 Phe 80 76 95 32 10'! 2.6. 112 133 22 111- 10 5 106 122 93 Ar; ' .119. .22 .122 .122 de 93 97 114 91 mm‘ 98 96 98 90 TAAf 26 g 105 90 cmOOII 3 m a! s 116083 “ n ‘ 1. Index ,3 PAA concentration from saline treated rats 1 100 bloweet index indicates the predicted limiting amino acid cThe limiting amino acid as determined by rat growth response or in theoretical calculation is underlined (Rae _e_t a}, . 1964) dTotal essential amino acids °Tota1 non-essential amino acids {Total amino acids 43 leucine in min. The PAAI method correctly identified the "known" limiting amino acid in only the soy diet. The PAAI method appeared to give low index values for isoleucino, leucine and pherqlalanine regardless of the protein fed. This obser- vation is supported by data recalculated from other studies (Munro and ‘l'hanpson, 19533 Crofford gt $1., 1964 and Potter _e_t_ _a_1_.. 1968 and the first experiment). An attempt to correct this "bias", which the PAAI had toward certain amino acids. was made by expressing the PAAI from each of the casein, soy and sein fed rats as a percent of the PAAI from egg albumin fed-rats. Egg albumin PAAI were used for reference because egg is the protein which supports optimal growth in rats and since whole egg PAAI were not available. Values expressed in this manner are shown in Table 14. Methionine was the limiting amino acid in both the soy and casein fed rats. While these were correct identifications (agreed with growth studies by Russel _e_t 11.. 1946), this procedure did not identify lysine as the limiting amino acid in coin. The failure of this procedure to identify lysine as the limiting amino acid in sein may have been the result of the catabolic state of these rats since they lost 50 gm (body weight) in the 2 week feeding period. When PAAI fraa sheep in experiment one were expressed as a percent of the egg albumin PAAI (calculated in this experiment). methionine was the first limiting amino acid whereas lysine and arginine were the next limiting amino acids on the low and high glucose treatments. respectively. This prediction agrees with other reports (Bergen _e_t $1.. 19683 Rois e_t_ 3.1.. 1969 and Conrad 33 3.1., 1968) which suggested methio- nine as the limiting amino acid of ruminants. Since dividing PAAI nu Table 115 PAAI Divided by Egg Albumin PAAI Experiment Two acid Val Met 11. Lou 'ryr Phe Lye His ££s__ ___[ fitnenflgp Experiment two Experiment one Low High Casein Sgy Zein Saline glucose glucose 96‘ 97 82b 86 77 7h 95 116 124 106 89 82 61‘; 2.3." 92 72" on” 51" 107 136 122 121 92 71} 91 116 101 11k 87 67 122 127 101 106 85 74 95 119 103 111 95 85 117 139 19.2 91 82 57 101 116 89 81 77 75 50 117 _9t 81 66 59 A blowest index indicates the predicted limiting amino acid °The limiting amino acid as determined by rat growth responses or in theoretical calculation is underlined (Rae g_t_ a_l_._. , 196“) ex I PAAI for other prgtein-fed rats PAAI for egg-albumin-fed rats x 100 45 from test proteins by PAAI frm egg albumin resulted in a higher percentage of correct predictions of the limiting amino acid (compared to the PAAI method by Potter _e_t §_l_. . 1968) it seemed worthwhile to further investigate this procedure. There was also the possibility that using whole egg PAAI. instead of egg albumin PAAI as a reference might increase the accuracy of this procedure since whole egg is of higher quality. Experiment Three This experiment was designed to determine the accuracy of limiting amino acid predictions which express PAAI fras rats fed low quality proteins as a percent of PAAI from aninals fed high quality protein (whole egg). Mean PAA concentrations of rats fed each of the eight protein diets are shown in Table 15. The plasna levels of all amino acids except aspartic acid and glutamic acid differed significantly (P < .05) with diet. No significant correlations were found between the EAA canposition of the diet and the plasma EAA level pattern of rats fed that diet. A second set of correlations were calculated be- tween the level of each amino acid in the plasma of rats fed the eight diets (acrossed diets) and the levels of that amino acid in those diets. Significant (P < .05) correlation coefficients were found for only methionine (r = .81) and lysine (r = .71). These significant correla- tions may be attributed to two things: first, methionine and lysine were the limiting amino acids in several of the diets and second, the dietary supplementation of methionine and lysine increased the plasma levels of these amino acids. The rats in this experiment had higher absolute PAA levels than the rats in experiment two. The average total plasma EAA level for endow cease Haven! .30.. ofiie Heap—330:5: .239: nodes and: Hans»: Havens. eosefiuap no eflahHass he nesfiuuevunn Go. v n: easel—ohm orange-.3 583.33. . Eco .“pflflnmeMfificdflimfi 3 HI ooa\ww as nee-sauna one use even 0H non onshore one one eesaasm . . . Exam and: 8.1% 8.3 own? :44“... who“ minim” mhw 3.3 .3 menmm mmnmw sewn idem. e Hel ‘Wfl ”H %m ”H “H 'HMUH 3‘ ”H ‘me a .3” m” N WWW” m m mmum seems omowos $5 “fun c. ”n m cum wo~.o M .N. mm .v m N m cm? 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Isa no 835588 was: ma canes #7 the rats in nu. experiment was 2a. 55 mg/lOO .1. whereas it was 13.82 mg/100 ml for the rats in experiment two. likewise. the PAA levels for casein fed rats in this experiment were double those in experiment two. The higher PAA levels, in this experiment may be due to the shorter fasting period before blood was collected (8 hours vs. 16 hours in experiment two). In comparison to the saline infused rats, the glucose infused rats had significantly (P < .05) lower plasma levels of all amino acids except alanine, cystine and ornithine (Table 16.) The effect of glucose on the PAA levels of each dietary group are shown in Appendix III. while the PAAI calculated from these values appear in Table 17. The PAAI procedure of determining the limiting amino acid (lowest PAAI) failed to identify the recognised limiting amino acids in the diets except for diet 2 where either leucine or valine was predicted as limiting. Fbrthermore, when the PAAI from rats fed the other diets were expressed as a percent of the PAAI for either whole egg- or casein-fed rats the recognised limiting amino acid was not predicted (Table 18). The recognized limiting amino acids are those determined by growth response of rats (Mitchell, 1959: HcLaughlan 23 51.. 1967 and Mitchell and Smutts, 1932 and Russel at 51.. 19%). Results of these experiments indicate that neither the PAAI method nor the procedure using a ratio of PAAI (PAAI from rats fed test proteins divided by PAAI from rats fed high quality proteins) are uaeable for determination of the limiting amino acid of the diet. Since the dietary limiting amino acids appeared to be reflected by a low level in the plasn, PAA concentrations were again used to calculate another index which might identify the limiting amino acid. The PAA concentrations of rats fed each test diet in experiment three 43 Table 16 Hoan‘ Concentrations of Plane. Amino Acids According to Saline or Glucose Treatment and Overall PAAI. Experiment Three Treatment Amino Significance b acid Saline Glucose $34 of I" value PAAI Essential amino acids m- 6.15 5.22 3.23 P <.01 85 v.1 2.33 1.83 35.08 P <.001 79 mt 1.02 .69 -00“ P <.001 68 11. 1.19 .91 Lou P <.001 76 Lou 1.86 1.27 $.12 P <.001 68 Tyr 2.01: 1.58 35.08 P <.001 77 Phe 1.06 .811 ~5.02; P .001 79 lys 6.72 5.77 1.20 P .001 86 His 1.2”: 1.12 1.04 P <.05 90 Arg . 2. 2.13 P .001 £523. mu" 227507” . Av. PAAI 79 Non-essential amino acids ‘8? .66 .53 3.03 P < .005 80 s.r £1.31 3.75 $.13 P .005 87 Asn 1.21 .92 $.05 P <.001 76 on. 6.21. 5.50 £2.25 P .05 88 Pro 3.08 2.45 t.1o P .001 80 Gly 2.38 2.19 1.07 P .05 90 Ala 5.97 5.09 —.1# N.S. 93 Cys .29 .110 $.02 ms. 103 am 1. 1.gz -.05 ms. 88 mm“ 25.19 22.05 Av. mun-8'7 TAA’ J2.26 “.312 ____ Av. TAAI 83 °Tota1 amino acids ‘Hean of #0 rats on 8 different diets and expressed as mg/100 ml bPAAI are glucose-group values divided by saline-group values x 100 °Tetal essential amino acids dTotal non-essential amino acids 1.9 Table 17 PAAI for Dietary Treatment Groups , Experiment Three “iota; Treahaent ou WK 4- Soy + Soy + soTy‘v “12°? 2‘ “1‘ ‘33” 3;” 2‘ $3 3‘ Thr 89'l 91 72 103 81 63 v.1 91 §2o 71 76 85 65 £1 83 Hot 71 98 _6_8_ 63 _1_<_)_9_ 51b 68" 57b 1.. 77 84 66b 71 83 71 87 80 Leu 61b 55b 67 57 87 72 79 81 Tyr 72 10 5 81 61b 79b 70 77 78 Phe 64 82 83 72 89 65 88 2g Lye §_2_ 109 78 18 98 g 83 83 H1. 79 101 81 101 88 86 86 90 be $2 .192 3.3.5 _§.9. .53! .92. .29. .21 Av. EAAI 76 91 75 78 87 70 83 82 Ave TAAI 87 _90 81 '82 83 26 8% 85 gm“ values a _concen on rem g cose s group x 100 PAA concentration from saline infused group bLowest index indicates the predicted limiting amino acid oThe limiting amino acid as determined by rat growth response or in theoretical calculation is underlined (Rae £0 _a_2_|._., 1961!) 50 Table 18 PAAI fru Rats Fed Other Diets as a Percent of PAAI fr- lute Fed Hhole Egg or Casein Diets _w— Expressed as a_percenrt_oTwhoIe egg Pm Die treatment Amino fi+AI 0.. can Soy Soy+AA Soy-r11 's‘oy-+"AA acid 2 L h L 6 *1 8 Thr 102 81.b 116b 91b 71 109 109 V91 22. 78 89 93 711, no 91b Hot 127 88 92 13% 66 88 7a 1.. 109 8'6 92 10 92 113 1011 1.. 0b 110 93 1113 118 130 133 m- 1 113 85 110 97 107 108 Phe 128 130 113 139 102 138 % Lys 176 126 1% 152 _1_2_6_ 13b 1 His 128 10 3 12 m 109 109 111+ Arg 12 3 101 95 100 98 90 87 _fi messed as a E! ent of casein PA:AI_I i _ ta _L Die treatmen ‘ Aline WE WE-l-AI CGH Soy Scyzn Soy-PAA Soy +1: 1 5 1 acid 2 it 8 __ Thr 124 126 1113 113 88 135 13 5 17.1 128 120 107 120 12 117 m _113 THE 93 2% 35" 10% 84" 11. 117 127ID 108 12 108 132 121 1... 91 82_ 8 130b 107 118 121 171‘ 89b 130 75 98 86 95 96 Phe 77 99 87 107 78 106 %l£ Lys 2% 1110 126 121 100 106 His 9 125 1‘2? 109 106 1°61. 111 L1!— 99_ 121 j“ 92 96 93 A 86 ‘Dietary treatments corresponds to diets in Table 1. bLowest no.2 indicates 1:11. predicted limiting amino acid °The limiting amino acid as detereined by rat growth response or in theoretical calculation is underlined (Rae _e_t_ ;_1_. . 1964) 51 were expressed as a percent of ”reference PAA concentrations”. The "reference PAA concentrations“ were obtained by averaging the PAA concentrations of rats fed whole egg, whole egg plus amino acid and casein since these diets supported excellent growth. The values ex- pressed in this manner are referred to as "plasma amino acid reference indices”. The reference PAA concentrations and PAA reference indices for experiment three are shown in Table 19. The lowest PAA reference index did correspond to the recognised limiting amino acid of diets 1, 2, 3, ll», 5, 6 and 7. The limiting amino acids were those determined in growth trials with rats, or by calculation (percent in diet divided by requirement). The reference index of diet 8 was lowest for leucine. The limiting amino acid in diet 8 has not been determined by growth trial but was calculated as being pherwlalanine. The reference PAA concentrations obtained in experiment three were used as a reference to calculate the reference indices for ex- periments l and 2 (Table 20). The expected limiting amino acid was predicted (lowest index) for 3 of the h diets of experiment two. Similarly when PAA reference indices were calculated from Bergen _e_t_ 2.3:. (1968) data, the limiting amino acids were identified in 2 of 3 cases. While the limiting amino acid is not known for the sheep in experiment 1, the PAA reference indices predicted lysine. This result must be viewed with caution as the reference concentrations were obtained from rats, not sheep. §§periment Four kperiment four was designed to evaluate the use of PAA concentra- tions in determining the limiting amino acid in proteins reaching the 52 Table 19 Plasma Amino Acid Reference Indices‘ for Ehch Dietary Treatment Group, Earperiment Three A _‘A _— Dietag Eodps HE + _ Soy 4- Soy + Soy—+ Amino concentration WE AA Cas CGH Soy AA AA AA acid 100 ml 1 2 3 1+ 5 6 7 8 Thr . 7O 87 138 1146 131 13 5 115 88 1.17— v.1 2.35 77 66 117 76 82 109 z_1_ 100 Net .86 65 106 _1_c_)_2_ 66 51 147 116 141 Ile l. 15 72 68 119 75 98 108 90 100 Lou 1.148 71 5; 122 203 95 116 89 95 Tyr l. 80 50 96 135 92 108 120 109 95 Phe .89 73 82 111 116 106 141 113 110 Iys 7.11 26 110 1115 22 97 fl 99 108 His 1. ll 72 100 113 13 5 123 98 89 103 4.2L $72.16 90 51 195 1.12 196 19», 181 206 w _i m ww— ‘Index values I PAA conc. for the saline infused rats fed test gotein diets b Réference PAA concentration x 100 Reference concentration refers to the average PAA concentration of the saline treated rats on diets l, 2 and 3 °Lowest index indicates the predicted limiting amino acid dThe limiting amino acid as determined by rat growth response or in theoretical calculation is underlined (Rae _e_t 51.. 1961+) 53 «8395:3200 44m ooaoaohom 38% :fl Mn 35 8.32.93 on 533933 Heodaoaoen» 5 no encodes.» 5.8km pen .3 pecan—woven on pace 2.33.- mfivfinfin one»... 3.. one... menses: 83.898 .5. .3385 885 8....ch ooa H town: pace—veep» Hoe.“ conveniences 44m w 03”; HobcHo Wm! m3 8 18a 3 «9 we 69 «mm 2. in on two. 2. so we on .8 new one 3 .2 mm 8 8 Ho .3 on. mma mm m3 we we mm em 2m an «NH 3 8: new mm mm nm 5 SH mm.” mm mm Q. we no we :3 ed” :3 we m3” mm R we we ed” 3 .3. one. Q. an em. om. mm c... mun m3 R 3 R R on an d5 3 on: mm m3 8 S R . Hm ha. and... 58.8 H323. do. fion new 538 eased. 3.. seem 13.0.5 mum and: 3 $36 .d. a. 4.an o5 ccoawnodé 11 wagon—dag .350 no.“ veadwheveo essence—H 3:98qu 304 end—.4 «Bee on sands 5" 5393:0080 44m eoaeaouem go: ON eHnee 5" small intestine of sheep. Mean PAA concentrations of sheep are shown in Tables 21, 22, 23 and 24 according to duodenal infusion treatment. Tables 21 and 22 show the pro-glucose (To) and one hour post-glucose (T1) PM concentrations on day 1+ of the protein infusions while Tables 23 and 24 show the To and T1 PM concentrations from day 6 of the infusion, respectively. The T0 PM concentrations from days it and 6 of the infusion were averaged as were the T1 PM concentrations from days 4 and 6, these appear in Appendix IV. PAAI calculated from the average PM concentrations of days h and 6 are shown in Table 25. These PAAI failed to identify the ex- pected limiting amino acids in the protein infusions except in the case of the soy plus methionine infusion (infusion 5). PM reference indices were calculated for each of the infusion treatments by ex- pressing To PM levels as a percent of reference PAA concentrations. Reference PM concentrations were calculated by averaging the To P“ concentrations from sheep being infused with whole egg and casein. Both the reference PM concentrations and PH reference indices are shown in Table 26. The lowest reference indices for the whole egg and casein infused sheep corresponded to threonine and histidine res- pectively. While neither of these amino acids are the limiting amino acids of those proteins, these results were not unexpected as both whole egg and casein are of high quality and both were used in calculating the PM reference concentrations. The PM reference index predicted nethionine and lysine as the limiting amino acids in soy and corn gluten neal, respectively. These results agree with growth trial re- sults indicating these as the limiting amino acids. Calculation of theoretical limiting amino acids by dividing the anino acid composition of the protein by the amino acid requirements 55 .36.. end!- Hopes." egos. and!» Henpsgeousos H333 938.. endl- Heflneeeo Hen—ea... Go. v a: 395.3 26803055 handofloflfle .- Eon «Eggheads add—”Emu 5.? sends?“ . 5:333.» on» own.“ pong our no€-§3ohm e5. 0» uneven: Ha cod? no 603893 one sea—”£5 neon pines £309.93 5.? coeds—H 53338 meenm new wastage 41m 3 hams...» ssfi .useiyeeua-eem wash 3 .efluevoum S 3%.... nine «NAN RAH Imm.m~ Horn Meta a.ma Mama H44% 0 e H o o e on Hod—H o 0 3a 6.: no.“ «mum! muml th: wmll mil mm"... mmql Muesli a use E 3 Mm E Mm n as n O 0' QC 0 do 0 80° C O O .mmn v m m.“ 3&2”. 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R; 3a RA .5 .m... o a mm. 8. 84 SA 3.." as. 84 an. 3m .m... ma.“ 8. 3.." S. a. No; R. an; 3. .5. .m... n3" 3. mm. SJ 3.. 34 SA 84 3. a: a... . w. .m. .w... ... W... m. mm. a. m. 0 0|. 3 o 0 o :0 o O o o o .3. a.“ n8; «3 .35 «3. .23 as; #3 a. g. 3 .3. 8.“ G. 3. cm. H. mm. mm. 8. an. ha. I :1 Hi .32. 2a.: Hqflatau f and.» m «a E» o W 0 ml 3 n N a 33 .8853 Min: H“. m: M pox + WM :8 mm awon mm 8.34 59 Table 2 5 Plum Amino Acid Indiccs‘ for Experiment Four Infusion:b Alina NE 00.8 Soy CG)! Soy Met EA WE 2X NE 31 acid 1 2 j __ L 5 6 7_ 8 Essentiul amino 1:213: m- 80 88 79 87 2° 13; 77 87 V0.1 62 76 73 85 3 211 62 611 Hot 72 1%; 3° 76 87 189 85 81 Il- 45° 94 3“ 283 59 “5° Ian I’16 58° 66 57° #3 33 38° 83 111‘ 7“ 109 81 73 39 191 78 77 Pb. 74 101 87 79 56 169 99 80 m 28: 129 88 131 88 22“ .112. 122 His 129 88 7 73 95 1:1“ 129 73 &-%%%§-§ % %25 73 81: Non-9889;111:131 uino acids 3» 88 93 92 83 58 67 66 7b Glu 108 76 111 113 69 11h 51 61 61: 93 91 83 98 63 7b 82 92 A1. 101 90 81+ 114 74 98 77 fig Cys 0 108 88 0 1 mm‘ “a! ‘8; ‘8'9‘ ib‘o’ ‘EE ‘3; 7% ‘85 1.118 _82 82 82 _90 61 121 71 _7_e_ :1 I! Post-ffufient “Inca“! PAA concentrati on b c“ B Pro-trutaent glucose concentrati; x 100 Refers to protein infused °Lovest index indiutcc the predicted uniting uino ccid d'l'hc limiting amino ccid u detonixud by at grow: response or m thcorotical calculation is underlined (RIO at. 1]... 1964) °Toul cssmthl amino acids 1.‘I'cflal nm-oascntinl amino acids 8Total ulna ucids 60 of swine (Table 27), were made for lack of a better comparison. This comparison can be criticized because the anino acids in a protein which are available for utilisation are not always the same as the amino acid composition. Threenine had the lowest PM reference index in sheep infused with soy plus methionine. According to the calculation in Table 27 threonine was the second amino acid in soy. Thus, the PM reference index prediction of threonine was correct as the first liniting amino acid, methionine, had been added to the diet in amounts so that the final methionine concentration in the diet was above the requirement. The theoretical limiting anino acid for egg albumin is either threonine or lysine. The PAA reference index was lowest for threonine. The other tire infusions were both whole egg, but differed from the first whole egg infusion as the amount of protein infused was 2 and 3 fold greater. The lowest PAA reference index corresponded to lysine for both the whole egg 21 and 3X infusions. Theoretically, methionine is the limiting amino acid of egg. However, if cystine fulfills W of the methionine require-ent (NRC Nutrient Requirement of Swine, l968) then lysine should be the limiting amino acid in whole egg. Further- more, lysine was shown to be the limiting amino acid in whole egg growth studies (Mitchell, 1959). Thus, these results indicate that the limiting amino acid of the protein reaching the snall intestine can be detenined by expressing PAA concentrations as a percent of reference PM levels which are obtained after duodemlJy infusing a high quality protein. To evaluate further the PM reference indices, plasma amino acid concentrations of conventional ruminants in other studies (without ducdmal re-entrant cannulas) were expressed as a percent of the 61 Table 26 PM Referenced Indices‘ for mch Protein Infusion Treatnent in Experinent Four JFFotein £53m" Aline “mm TE 21' us ‘33:" acid Avb (l + 2) '1 2 3__ h 5 6 7 8 m .65 71° 128 103 17!» 58° _22" 168 155 v.1 2.30 ' 81 ms 107 96 78 50 106 no Hot .39 93.1 3); 16° 97 388 121 29 151 11. 1.10 96 103 1.16 65 95 37 66 93 Leu 1. 76 117 132 119 172 72 21+ 1148 126 m- 1.06 79 120 90 185 72 31 136 167 m. 1.11 101 99 91 139 123 52 91 133 In 1.67 §_1_ 119 120 91° 163 51 g" 51° 31. .73 127 71" 182 153 90 1711 127 103 Arg 1.73 101 99 190 77 107 77 106 '15:: conc Em aninls on test gotein PM conc from animals on reference protein bAverage PIA concentrations from whole egg and casein infused sheep, pre-treatnent. Expressed as mg/lOO m1 °Lowest inlet indicates predicted limiting amino acid dThe limiting amino acid as determined by rat growth response or in theoretical calculation is underlined (Rae _e_t_. _a_._l_., 196“) aReferenced Indices = 62 Table 27 Theoretical Limiting Amino Acid of Proteins __ * Wotein _ Amino acid __ requiruent" Amino of swine Whole Egg acid A L of diet_ __ ‘ §L Casein egg albumin '11:: .115 8.7lad 10.0‘1 10.1; 9.3° Val . 50 10.4 1‘}.8 14.0 16.2 Met. . 50 2.2" 6.6° 8.0° 9.8 II. .50 11.6 13.2 12.8 13.6 Leu .60 12.6 16.8 15.0 15.0 Phe: .50 9.6 11.6 12.0 111.2 IV! 070 90“ 1107 901d 903° His .18 13.9 16.7 13.9 12.2 A11: :20 35.0 21.5 29.5 30.0 ‘nke; from u.n.c. Nutrient Requirements of Swine 1968 b ' Percen_t_amino acid in otein Detezmined ‘8 “11"” Mo acid W °Indicates first limiting amino acid dIndicates second limiting amino acid 'Does not account for cystine being able to fulfill 40% of the methionine requirement fDoes not account for tyrosine being able to fulfill 30% of the phenlalanine requirement 63 Table 28 PM Reference Indices.‘ of Conventional Sheep _firser et 93;. fitjen et al. Ely et al. (12667 412667" 51262 Amino acid A B C D B faun B def S Urea Zein "m—W“"”“302 — ._...._.__ 197.36? +—W""‘Tfi—‘2 v.1 g9 76 13413 126 113 zggb Z 5 '59 28 m 5 __ 3 n: 68 g 15% 91 7 156 7% 6% 63‘“ Leu 75 76 66 60 72 139 51 91 127 m 167 109b - - 78 198 23 21b 97 an 79 66 12 .99. g 11.1 36" 30 75 Lys 1%: 122 131 __ 161' 60 1:6 135 His 2 2111 195 119 5.8 64 78 391 Are 39 79 - - 77 161 55 M 68 Potter et 91. Schelling _e_t_ _a__];. 119657 11967) Amino acid mpt one Glu To Glu 6 hr Purified SH! Thr 285 351 232 17" 35“ Val 131 96 70 M 185 ' not .122 10.6. 71 22" 62" 11. 121 103 83 39 137$ Lou 98 100 76 30 206 1‘1? 98b Ebb 97 61 278 Phe QB §_l_ ab 39 1166 Lys _11__ % 81 1+8 129 His 107 13'3“ 104 127 £5_- 112 - - 116 19 ‘ ‘ih’conc EW ‘RefOMOOd 1nd“ a PAL cone frm whole egg and casein infused sheep bDesignates the limiting amino acid as determined by the proposed reference index method °The theoretical limiting anino acid(s) are underlined. These estintes are based upon the amino acid composition of the diet and upon the amino acid composition of rumen bacteria and are thus somewhat arbitrary 6“ reference PM concentrations determined in this study. The PM reference indices predicted fran these studies showed pherwlalanine (6 times), methionine (6 times), lysine (once) and isoleucino (once) as the amino acids limiting in the protein reaching the small intestine of the ruminant (Table 28). In the study by Ely gt 5;. (1969) lysine was expected as the limiting amino acid, since a sein diet was fed and zein is deficient in lysine. In the study by Schelling _e_t_ 3;]... (1967) methionine was expected limiting as rumen bacterial protein and soy protein are methionine deficient. The reference index correctly predicted methionine in both cases. The limiting amino acids in the other studies may have been methionine or pherwlalanine, as bacterial protein is deficient in methionine and phenylalanine. However, this does not preclude other amino acids from being limiting to the sheep. Experiment Five This experiment, using sheep, was designed to determine whether the amino acid levels in jugular vein blood differed from levels in blood frau the carotid artery, both before and one hour after the intravenous infusion of either saline, glucose or acetate. The mean concentration of venous and arterial PAA are shown for each sample time (pro-treatment ”To" and post-treatment ”Tl") in Table 29. There were no differences between the venous and arterial PM levels at either T0 or T . At T 0 the total EM concentrations were 9.80 and 9.99 l mg/100 ml for venous and arterial blood respectively, whereas the T1 concentrations were 7.81 and 8.05 mg/lOOml for venous and arterial samples, respectively. The venous and arterial PM levels paralleled each other as they decreased from To to T1. The mean To and T1 PM concentrations for each infusion treatment 65 Table 29 Mean Concentration‘ of Venous and Arterial Plasma Amino Acids Before and (he Hour After Treatment, Experiment Five _— _ns. ‘filire-treatm—ent (To) ‘ :Post-treatnent TTQ Amino Sam 1e siteb Sam le site acid __ Venous Eterial SEH Venous Arterial SDI Essential amino acids m 1.08 1.03 .18 .79 .83 $.18 Val 1.69 1.82 $.21 1.110 1.116 $.26 Hot .23 .21; $.03 .19 .20 $.02 Ile .88 .92 $.15 . 59 .65 $.17 Leu 1.50 1.56 1.25 1.01 1.15 $.30 Tyr e8]- e8“ te1]- e68 e69 tea? 5. e65 e63 tall 052 053 i007 If! .8]. e87 ie29 e69 e70 i317 His .63 .6“: fig .28 .252 $.15 ‘rg 1 1. 0 1e 10 £035 mu" 9.30: 9.99 7 .81 8'. '0' '5' Non-essential amino acids up .26 .21; $.07 .20 .20 $.03 301' e99 e97 i025 e89 e89 32.2“ Glu 3.13 3.19 $.91 2.81 2.80 $.83 Ala 3.03 2.77 $.25 3.06 2.84 $.59 Pro 1.20 1.00 $.12 .89 .22 211.19 Cys . 2 .17 . . 2 $.12 TNEMd "8"8'. 3' “8“2‘2' 9 T2: 20 "8—”. 07 Tu' , 18 .63 18.68 _ 16 . 01 16 .312 ‘Values are expressed as mg/loo m1 bSample site refers to either jugular vein or carotid artery cTotal essential amino acid d'l‘otal non-essential anino acid .Total amino acid 66 are shown in Table 30. The T0 methionine and tyrosine concentrations in the acetate infusion group were significantly (P <.05) higher than the methionine and tyrosine concentrations in the saline and glucose infusion groups. The T1 plasma tyrosine concentration in the acetate infused group was significantly (P (.05) higher than the concentration in the saline and glucose infused groups. The T1 plam methionine concentration after glucose infusion was significantly (P (.05) lower than the methionine levels in the saline and acetate infused sheep. The effects of the infusion treatments upon the combined venous and arterial PM levels are shown in Table 313 first by expressing the T1 PM as a percent of the TO concentrations (PAAI) and then as absolute PM concentration differences (To-T1). The average essential amino acid PAAI are 90, 80 and 73 for the saline, glucose and acetate infusion groups. Potter _e_t a_l__. (1968) infused both glucose and acetate into the carotid artery of sheep and found lower PAAI in the glucose infused sheep. The results from these two experiments do not agree; such differences may be due to the small numbers of sheep used in this experiment or to different infusion sites (venous in this experiment vs. arterial in the 1968 study). A notable difference between the glucose and acetate treatments is that the acetate infusion resulted in a sizable decrease in the plasma non-essential amino acids levels whereas no decrease was noted after glucose infusion. This suggests that there was little non-essential amino acid synthesis when acetate was infused. 67 ages canals Haves amass oudls Hununeeeeuncn. H395 nodes cad-3 Hefivneene Haves“ .Ano.v my ease.» edmflflen 3:355 $433.3 esonemoaon Handbags .- 3.83.5 unsaved Side .3133 so eeooeam .enflnee segue no son—fleas.“ e5 3 enema." #805356 7 HI coin: as teenage 8.3 amid 5.3 mime 3.5 3.3 3. EB: more. mud NEH «m4. mm. was. omen 8.“ HM. N3. is ems: NHeIH NW. e 3e 5 6.2 3....” 36. «TN and 89v a 3.“ nmma send ommé 34 .mi 3...... no.3 R. S. 38.v a 84. pan; «8. one. 2a .n.z on.“ 8.~ ohm 8.~ 6.2 so.“ 2k 85 luau 3a .m... an.“ on. a. a. .n.z 3.1:. 3.3 an. R. you .m... no.“ .8. 3. om. .mi no.“ mm. mm. cm. a: .30.. Odd... 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GENERAL DISCUSSION Protein synthesis is influenced by both amino acid and energy supplies to the cell. The quantity and quality (distribution) of amino acids supplied is a major factor determining rate of protein synthesis (Munro, 1968a). While withdrawal of protein from the diet of an animal decreased the stability of microsomal protein synthesizing machinery in the cell (Munro 1968b, 1969), increases in the liver amino acid levels increased polyscme formation (Munro 1968a). The rate of sulfur amino acid incorporation into protein was directly correlated to RNA concen- trations in liver, kidney and muscle (Allison _e_t 93;" 1963). Energy, (ATP) is another main factor affecting protein synthesis (Munro _e_t_ a_2_l._., 1962). Protein synthesis appeared to account for decreases in plasma essential amino acid levels when glucose (substrate for ATP) was given to semi-fasted animals. The pattern of decrease in the concentration of the plasma EM (decrease in each essential amino acid) resulting from glucose ingestion or infusion was correlated to the m caaposition of striated muscle (Munro and Thompson 1953 and Potter _e_t_ _a_l_., 1968). Blood amino acid levels are a reflection of the larger tissue free amino acid pool (Rogers and Harper, 1968). Since the concentrations of plasma and tissue free amino acids are influenced by digestibility, quantity and quality of the dietary protein, quantity and type of energ substrates in the diet and rate of cell protein synthesis, then limiting amino acid determinations based upon plasma amino acid concentrations will be influenced by these factors. 69 70 McLaughlan and Morrison (1968) emphasised the importance of reference PAA patterns when using PAA concentrations to determine the limiting amino acid. The methods of McLaughlan (1964) and Longenecker and Hause (1959) used fasting PAA levels as a reference, while Hill and Olsen (1963) used the PAA levels after a non-protein diet was fed. Reference PAA concentration patterns were used as a reference point to compare subsequent changes in PAA concentrations which occurred when test proteins were fed. Potter _e_t_ _a_l_. (1968) used 24 hour ”fasting" plasma amino acid concentrations as a reference point in studies with sheep. This 24 hour post-feeding sample was expected to represent the time at which energy supply dictated neither an anabolic or catabolic state and a time when protein supply was neither minimum or maximum in comparison to other post-feeding times. The limiting amino acid determination method (Plasma Amino Acid Indices) reported by Potter e_t_ 9.3:. (1968) was based upon the following theoretical assumptions; that glucose infusion would provide more ATP, the presence of more ATP would stimulate protein synthesis and protein synthesis would withdraw free amino acids from the plasm and tissue pools as they were needed. Since the limiting amino acid is the amino acid in shortest supply relative to the metabolic need, then expressing the post-glucose PAA levels as a percent of the pro-glucose PAA levels (PAAI) will predict the limiting amino acid (lowest PAAI). The results of experiments 2 and 3 with rats and experiment 1+ with duodenally cannul- ated sheep showed that the PAAI method does not predict the dietary limiting amino acid as was previously established in growth trials. Therefore, it is unlikely that the PAAI method (Potter e_t_ _a__l_. . 1968) can identify the limiting amino acid in the protein reaching the duode- num of sheep. 71‘ The ELAIJmethed assumed that glucose would stimulate protein synthesis and that the amino‘acid requirement for protein synthesis was equivalent to the total amino acid requirements of the animal. The results of these experiments suggest that the EIAI method does not accur- ately predict the limiting amino acid even though glucose induced protein synthesis. Thus it appears that the EIAI procedure will not identify the limiting amino acid because aminomacid requirements for growth and.main- tenance differ. .An example of'an amino acid whose maintenance require- ment differs from the requirement for protein synthesis is methionine. Methionine (S-adenosylmethionime) serves as a methyl donor in the synthe- sis of phosphatidyl choline from phosphatidyl.othanolanine. Thus the methionine requirement will differ in respect to the rate of lipogemesis in the liver (synthesis of phosphatidyl choline). Furthermore the meth- ionine requirement is somewhat undefined as cystine can fulfill part of the methionine requirement. Likewise, tyrosine can replace part of the phenylalanine requirement. Thus, the EIAI procedure may have identified the limiting amino acid for protein synthesis, or at least the limiting amino acid for protein synthesis in.a specific organ (Munro 1969) and may explain why the EIAI procedure did not identify the amino acid which was limiting in growth trials. u Since EIAI for leucine, isoleucine and phenylalanine in experiments 1, 2... 3, were unduely low, the PAAI from rats fed poor quality diets ‘were expressed as a percent of EIAI from rats fed high quality proteins. This procedure was expected.to.remove the.'bias" effect of glucose on specific amino acids and allow determination of the limiting amino acids. This procedure failed to identify the limiting amino acid when PAAI from rats fed different proteins were compared to reference EIAI from rats fed either whole egg or casein. 72 Sevenl research groups (sanerIiCh ‘nd Sahon 1955’ Grey 9}- El" 1960, Hill and Olsen, 1963 and McLaughlan and Morrison, 1968) reported low plasma levels of the limiting amino acid. Results from experiments 2 and 3 are in agreement with these reports, as the plasma level of the limiting amino acid was lower than levels of that amino acid in rats fed other proteins. In experiment 3, the plasma levels of lysine and methio- nine were correlated with the dietary levels of these amino acids (these were the limiting amino acids when these proteins were fed to rats). This suggested that the plasma level of the limiting amino acid is depen- dent upon the dietary level. Zimermn and Scott (196 5) reported low plasma levels of the limiting amino acid when the dietary level of that amino acid is kept below the requirement. In theory, an animal fed a high quality protein would not have a low plasma level of any one amino acid (either all low or all high), while a low quality protein would cause a low plasma level of the limiting amino acid. Consequently an animl should exhibit an "optimal" PAA concentration pattern when fed a high quality protein. The limiting amino acid of the dietary protein should be the essential amino acid with the lowest per- centage value when the PAA levels of animals fed low quality proteins (less than ”optimal“ quality) are expressed as a percent of PAA concentra- tions from rats fed high quality proteins. These percent values were re- ferred to as “PAA reference indices" and the PAA concentrations from animals fed the high quality protein diets as "reference PAA concentra- tions." When PAA reference indices were calculated for the 8 protein diets in experiment 3, using reference PAA concentrations from rats fed whole egg, whole egg plus amino acids and casein, the lowest PAA refer- ence indices corresponded to the predicted amino acids shown to be limiting rat growth (Rae _e_t_ 11.. 1964) in 7 of these diets. 73 To apply the reference indices method (determination of the limit- ing amino acid) to sheep, reference PAA concentrations had to be deter- mined. Referenco PAA concentrations were obtained from sheep duodenally infused with whole egg and casein. These proteins may not give the 'optimal'.‘ reference concentrations but were used because they are high quality proteins. Crystalline amino acid infusions composed so that every amino acid is equally limiting should give the "optimal" reference PAA concentrations, but the composition of such a mixture for sheep is unknown. Reference indices calculated by expressing PAA concentration patterns from sheep infused with other protein as a percent of the whole egg and casein reference PAA concentration identified the known limiting amino acids in 6 of the 8 protein treatments. The reference PAA concentrations, as determined in sheep infused with whole egg and casein, were compared with PAA concentrations of conventional sheep. Results showed methionine, phenylalanine, lysine and leucine as the limiting amino acids in the different studies (Table 28). A hypothetical limiting acid was proposed for the animals in each of these studies by taking into account the dietary protein source and the amino acid composition of rumen microbial protein. The amino acid composition of the dietary protein was considered because some of the diet- ary protein will escape rumen degradation and reach the duodenum (Smith 1969). The theoretical limiting amino acid of rumen bacteria was deter- mined by dividing the amino acid composition of rumen bacteria (Purser and Buechler, 1966) by the essential amino acid requirements of swine (NRC Nutrient Requirement of Swine 1968) . This calculation showed methionine and phenylalanine as the first and second limiting amino acid of rumen microorganisms. 74 The diets fed by Purser _e_t_ al” (1966) contained corn, corn cabs and alfalfa meal. Diet A was high in corn, thus lysine which is limit- ing in corn may have been the limiting amino acid in the protein passing to the duodenum. In contrast diets B, C and D contained more alfalfa meal and less corn and corn cobs, thus very little dietary protein would have passed undegraded to the abomasum. Hence, the limiting amino acid(s) of bacterial protein were thus expected as limiting. Reference indices obtained using Purser's data with the reference PAA concentration from this study showed lysine, phenylalanine, methionine and methionine as the limiting amino acids on diets A, B, C and D respectively. Reference indices for faunated and defaunated sheep, fed diet B, showed methionine as the limiting amino acid in both faunated and defaunated sheep. Oltjen and Putman (1966), fed soy and urea diets to steers. Methio- nine might be the limiting amino acid in both of these diets as methionine limits rat growth in soy-fed rats and methionine is the limiting amino acid (theoretical calculation) of bacterial protein. Reference indices showed phervlalanine as the first limiting amino acid for both diets. This difference may be due to the fact that plasma amino acid concentra- tions from steers were compared to reference PAA concentrations from sheep. Furthermore, sheep are expected to have a higher methionine re- quirement than steers as wool contains fairly large quantities of sulfur amino acids. References indices from a similar study with sheep (Schelling _e_t_ al" 196?) shows methionine as limiting when soy and urea diets were fed. Ely _e_t_ _e_l. (1969) fed sheep a zein protein diet. Since sein is devoid of lysine, lysine was expected as the limiting amino acid. Refer- ence indices using data from these sheep did not show lysine as limiting. 75 “hen the reference plasma amino acid concentration pattern was compared with data from experiment one and data from Potter 933 9;]... (1968), phenylalanine was the limiting amino acid. The reference indices cal- culated from Potter gt a}, (1968) agrees with reference indices recal— culated from diet B, Purser _e_t_ 51. (1966), as both indicated pherwlala- nine as limiting (both diets were identical). The amino acids identified as limiting by the PAA reference index did in most instances agree with the estimates of theoretical limiting amino acids. Although unequivocal proof as to the accuracy of the PAA reference index procedure has not yet been attained, the reference index method.appears to be able to identify the limiting amino acid in pro- teins infused into the duodenum of Sheep. Proof as to the accuracy of this method will require further investigation in both rats and sheep. The final study of these experiments suggested that blood collected from either the jugular vein or carotid.artery would be of equal value for use in determining plasma amino acid concentrations. l. 2. 3. 7. 8. CGCLUSICNS Glucose infusion into the jugular vein of sheep or into the stomach of rats resulted in a decrease in the plasm amino acid concentrations. The plasma essential amino acid decrease pattern (decrease in each EM), which occurred after the infusion of glucose in sheep, was significantly (P <.05) correlated (r = .80) to the amino acid com- position of striated muscle of lambs. The plasma amino acid index method described by Potter e_t_ _a_l_._. (1968) did not identify the dietary limiting amino acid in rats. In rats, the limiting amino acid of dietary proteins was reflected by low levels of that amino acid in the plasn. Suppluentation of the limiting amino acid into the diets of rats at levels which exceed the animals requirement for that amino acid re- sulted in increases in the plasn level of that amino acid. The limiting amino acids of some dietary proteins can be determined by comparing the plans amino acid levels of rats fed those proteins with the plasma amino acid levels of rats fed a high quality protein diet (whole egg and casein). 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APPENDIX I SAMPLE CAICULATIQI 88 Appendix I Table 1 Sample Calculation Experiment One3 Effect of Glucose on PAA Isoleucino One Hour After Glucose Infusion Restricted Approximate Level d.f. Mean Square F of Significance Treatment 2 0.5491 18.723 P < 0.0005 Regression about Mean 19 0.0589 0.625 P < 0.837 Error 16 0,0943 Total 35 Unrestricted Approximate Lovel d.f. Mean Square F of Significance Regression about Mean 21 0.1056 3.60 P < 0.009 Error 14 0.0293 Total 35 .02 + Standard Error = 2 I: -.12 Duncan's New Multiple Range Test Critical Value (P < .05) .35 .37 P = 2 P 8 3 Ranked Means3 1.16 0.30 0.86 0.12 0.42“ 0.74 APPENDIX II PLASMA AMINO ACID CONCENTRATIONS FOR SALINE AND GLUCOSE TREATED RATS, EXPERIMENT TWO Table 13 Egg Albumin and Casein Diets Table 23 Say and Zein Diets 89 Appendix II Table 1 Mean.‘ PAA Concentration of Saline and Glucose Treated Hats for Each Dietary Treatment, Experiment Two __a_ Dietary treatments Egg albumin fiCasein Amino acid Saline Glucose Saline Glucose Essential amino acids Thr 1.36 —.16b 1.38 $.14 4 .34 3.36 4. 21 —.39 Val 1.32 _.05 1.12 £10 1.30 £04 1.05 I.10 Met .20 $.04 .23 i.03 .23 _.02 .16 ...03 Ile .64 1.015. .46 $.04 .62 _.02 481305 Leu 1.11 22.0 .84 i.11 1.11 _.03 .76 $.10 Tyr .49 4:. 02 .42 1.03 .55 + —.02 .58 ”5.05 . Phe .54 _.03 .43 £03 .55 _.02 .42 $.04 Lys 4 .45 $.30 4. 26+ -.50 4.58 1:.30 5.11 i.47 His .39 :03 .41 i. .03 .51 $.05 .54 $.05 Arg 1.03 08 1.1 .07 1.0 .10 1.02 .07 TEAAc 11.53 10.67 1 . 113.33 Non-essential amino acids Asp .90 35.08 .86 i.04 .78 L13 .63 L08 3..- 3.42 $.26 2.97 i.08 3.95 1.17 3.53 i=.25 Glu 3.23 1.19 3.09 $.29 3.17 $.21 2.40 $.13 Pro 1.45 1.08 1.57 1.09 1.66 1.10 1.57 1:.17 Gly 4.68 1.43 3.38 22.14 3.35 i.16 3.07 1.20 11. 3.49 1.15 4.86 12.37 3.24 .+..18 4.21 1412 Cys .33 to», .40 1:05 .38 L02 .40 t.03 0m d .45 1303 . 1 12.03 .44 i:.01 . 0 $.04 TNEM 17.95 17. 13.97 .29 TAA 29.48 28.31 31.81 30.62 "Mean of 6 rats expressed as mg/100 ml bStandard error °Total essential amino acids dTotal non-essential amino acids °Total amino acids Appendix II Table 2 Mean" PAA Concentration of Saline and Glucose Treated Rats for each Dietary Treatment. Experiment Two Diotgg treatments Soy Zein Amino acid Saline Glucose Saline Glucose Essgntial‘amino acids on 2.89 3122" 2.83 3.35 4.52 3.31 3.77 1.28 Vol .88 :t.05 .87 1.12 .80 1.09 .74 1.07 Met .15 1.01 .10 1.02 .32 1.05 .34 15.03 Ile .43 1.02 .42 £.07 .40 1.05 .35 1.04 Leu .75 1.04 .66 1.11 1.19 1.19 .92 1:.13 1371- .43 L03 .47 1.07 .55 1.10 .48 $.06 Phe .38 1.02 .36 $.05 .65 1.08 .53 i.06 Lys 3.97'i.89 5.05 i.87 3.29 1.36 3.27 $.68 His .54 1.06 .66 i.10 1.09 1.20 1.01 2.17 Arg c 1.12 1.09 1.45 1.14 _1.60 i.12 1.6 1.25 TEAA 11.36 12.92 5.51 13. Non-essential amino acids Asp .78 i205 .64 i.07 .80 t.04 .67 i.13 Ser 4.27 1.30 3.78 1.31 6.57 Me 5.38 1:.29 Glu 2.56 4.11 2.12 1.09 3.00 Jr..18 3.13 1.35 Pro 1.41 1:08 1.32 1.07 2.13 i.28 1.69 i.11 Gly 3.32 1.18 2.95 1.25 4.34 i..27 3.33 1.41 A14 2.55 1414 3.68 1.36 3.49 1.43 3.95 $.38 Cys .26 J$.03 .31 1.03 .24 i.03 .41 1.04 Orn .43 .03 .52 1.07 .8 5;..12 . 8 i.10 TNEAAd 15.58 15.31 21.42 19.34 TAA° 26._94 28.23 35.83 32.40 ‘Mean of 6 rats expressed as mg/100 ml bStandard error °Total essential amino acids d'l'otal non-essential amino acids ’Total amino acids APPENDIX III MEAN CONCENTRATIONS OF ESSB‘ITIAL AMINO ACIDS FOR SALINE AND GLUCOSE TREATED RATS, EXPERIMENT THREE g‘fi-‘L3‘5 ”ft-nu ' " ‘u-‘N . V' 3 Mean? Concentration of Plasma Essential.Amino Acids for Saline Treated Rats by Dietary Treatment, .Appendix III Table 1 91 Experiment Three 1 Dietary treatment group6 WE + Soy + Soy + Soy + Amino WE 4AA Cas CGMI Soy AA AA AA acid 1 2 3, 4 5g 6 7 8 Essential amino acids Thr 4.32 6.86 8.24 6.88 7.04 6.64 4.20 5.60 val 2.16 1.68 3.21 2.02 2.08 3.10 1.79 2.56 Met .63 . .92 1.04 .70 .44 1.66 1.19 1.55 150 .94 .85 1.65 1.00 1.24 1.44 1.11 1.27 Leu 1.30 .98 2.15 3.85 1.47 2.00 1.52 1.55 Tyr 1.03 1.68 2.69 2.07 2.17 2.55 2.22 1.93 Phe .79 .80 1.08 1.20 .99 1.53 1.08 1.01 Lys 2.17 7.58 11.60 1.43 7.10 7.92 7.70 8.42 His .85 1.10 1.39 1.49 1.45 1.18 1.07 1.20 Arg 2.12 1.95 2.40 2.68 4.59 4.61 4,36 .18 T ° 16. 31 24.40 35.45 23.32 28.57 “32.63 23124 30.27 Mean“ Concentration of Plasma Essential Amino Acids for Glucose Treated Rate by Dietary Treatment, Experiment Three Dietary_treatmentggpupE Amino acid 1 2 <3, 4 5 6 7__ 8 Essential.amino acids Thr 3.82 6.26 5.95 7.09 5.70 4.16 4.06 5.40 val 1.96 1.44 2.27 1.54 1.76 2.01 1.56 2.11 Net . .90 .70 . . .85 .80 .89 150 .72 .71 1.09 .71 1.03 1.03 .96 1.02 Leu .79 .53 1.45 2.18 1.29 1.44 1.20 1.25 Tyr .74 1.76 2.17 1.26 1.72 1.77 1.71 1. 5o Phe .51 .66 .90 .87 .88 1.00 .95 .95 Lys 1.35 8,24 9.07 1.40 6.64 6.07 6.39 6.95 His .67 1.11 1.12 1.50 1.27 1.01 .92 1.08 Arg c 1.79 2.00 2.05 2.14 3.87 .43gzz .44 3.80 TEAQ 12.84 23.59 26.77 19.13 24.60 23.11 21.99 2 .95 14.1 39.51 44.49 42.24 56.57 48.44 42.70 40.94 44.64 8Mean of 5 rats expressed as mg/100 ml bRefers to diets in Table 4 cTotalessential amino acids dTotal amino acids ['1‘ ' .J“%L‘ L «a?! '\P03AY ' - APPENDIX IV AVEAGE OF MY FWR AND MY SIX PLASMA AMINO ACID CQICHITRATIQIS IN SHEEP NODENALLY INFUSED WITH PROTEINS Table 13 Pro-treatment PAA Concentrations Table 2 3 Post-treatment PAA Concentrations 92 Appendix IV Table 1 Plasma Amino Acid Concentrations‘ of Sheep Duodenally Infused with Different Proteins, Ayerage of Days Four and Six, Pro-Treatment Experiment Four Infusion treatments” Soy 4- Amino WE Cas Soy CGM Met EA WE 2X WE BX acid 1 2 __3 4 <_5. 6 7 8 Essential.amino acids ‘— ___ Thr .146 .83 .67 1.13 o3]. o1“ 1.09 1o01 Val 1.87 2.72 2.46 2.21 1.79 1.15 2.43 2.53 Met o32 e35 o26 o33 1o32 ob]- o27 one Ile 1.06 1.13 1.28 .71 1.05 .41 .73 1.02 Len 1.51 2.00 1.80 2.59 1.09 .36 2.23 1.91 Tyr .84 1.27 .95 1.54 .76 .33 1.44 1.77 Phe 1.12 1.10 1.01 1.54 1.37 .58 1.01 1.48 Lys 1.35 1.98 2.01 .79 2.73 .86 .94 .96 His .93 .52 1.34 1.12 .66 1.27 .93 .75 Arg 1.1 pg}. 2. 2 1.34 1. _;:§ 1. 1.84 TEAAc 10.65 13. 5 13596 13.30 12786 7.35 12:41 13.75 Nonpessential amino acids Ser .66 .81 ' .92 1.x .40 .91 1.06 1.12 Glu 2.00 2.11 1.74 1.67 1.79 l. 67 1.57 1.98 313 4.27 4.91 5.80 7.76 3.55 5.32 5.95 6.2? Ale 1.23 1.75 1.7% 1.60 1.79 1.31 1.3: 1.86 Cys o o o3 o 1 osl o o o TN wmd "9". "0'3" 10 . 1 1 '.'54 12182 8.04 "9'.%E '1'0"."7'6 11.77 ff 19. 68 23.59 24.44 26.12 20.84 16.83 23.17 25.52 No. a Animls 5 4 l 4 l 3 l 1 aValues are expressed as mg/loo‘ml bRefers to different protein infusions °Tota1 essential amino acids dTotal non-essential.amino acids eTotal amino acids f Number of’animals per infusion 93 Appendix IV Table 2 F1asna.Anino Acid Concentrations‘ of Sheep Duodenally Infused.with Different Proteins, Average of Days Four and Six, Post-Treatment Experiment Four Infusion treatmentb Soy + Amino WE Cas qu CGM Met EA WE 2X NE 31 acid 1 2 4 5 6 7% 8 Essential amino acids Thr .37 .73 .53 .98 .10 .27 .84 .88 val 1.16 2.08 1.80 1.87 .77 2.43 1.51 1.63 Met .23 01+]- 019 025 10 15 061 023 039 Ile .48 .77 .79 .67 .36 1.16 .43 .46 Leu .70 1.15 1.18 1.47 .47 1.92 .77 .83 Tyr .62 1.38 .77 1.13 .30 .63 1.13 1.37 Phe .83 1.11 .88 1.21 .77 .98 1.00 1.19 Dys 1.24 2.55 1.77 1.21 1.32 .80 1.24 1.47 His ' .68 .67 .92 .97 .38 1.21 1.22 .97 .Arg 1.2 1.3 1. 1.02 1. 2 2.0 . 1.0 mu" "7'34 12.2% '1‘0‘.%2E' 10.73 "7714 210% ““9764 '1"0'."2‘!5$ Non-essential amino acids Ser .58 .75 .85 1.06' .23 .61 .70 .83 Glu 2.16 1.60 1.93 . 1.90 1.23 1.90 .80 1.21 01y 3.96 4.46 4.81 7.59 2.24 3.93 4.88 5.79 .Lla 1.67 1.2; 1.44 1.22 1.22 1.28 1.33 1.52 Cys e l a 0 9 o o a 2 a 2 2 e 2 TNEAAd 8.8'8' 8.02 9.42 12581;. 5.1% 8.24 7.98 9.68 TM° 16.22 21.94 20. 04 23.60 12.62 20.32 17.02 15.92 8‘Values are expressed as ng/100 n1 bRefers to different protein infusions cTotal essential amino acids dTotal non-essentia1.amino acids °Tota1.amino acids