" ..S 11 I; IIII‘gfiII '~ MI 1.; .“I' I . S ,. '»' . 1-."SH SJ S33; 13¢:th— . SS "‘."'”S“;{”.“S.S€I2I5. IISS..~.~ a, I S -’-.. ‘ 'II ' l'SSI' '1 3 S S" S (‘91-qu 'r‘ 3 “SSS S ‘ n‘a‘t'S . ‘. 7 SH ~I.S...I.ISSS:*.~IS . .- 5'”. HI: I.‘ .. .' ~. , 5"” ‘ II1S'S‘ ~t1'.t. “9-. . I5 1 I S" SISI‘IS“: SSSSSS Sui: SIriISIjI‘IS -\_ ISIHSIISI SIS IS I D 2—0 V {SSS . . . .3 ...-- _,._——__"-.. ::—-~- < . - A: 7:...f'fiv ,LS IS I S}; \ . {I .IIIIIIIII1 ISIS ., 'SIS‘S * . SSS“ 'SSSI'S ‘S S J. :. S. i. ..-. '. v‘ SIS??? * ISIS I I w - . ~~S - I :z‘ : SS \ 1| (SE' IS‘fiIII {Iggy S SS: .w .1 - O F'..._\— . ' .--v_'.- \’ w ‘— . F. , I. ‘ . r -A ‘ .a"“ . f 3.; Q '— . "Est", " virg— ‘Ha. —~._'5:5.2r:hr - .—- #5:; ., . : .—.» ‘,_.-fi_ . ' - ' c ”’53. -‘ °’. , .— we r. . _ 4 “a . - a. w _ 40W , 1%,. 5m RISES-SSS. St S'Stv‘ \ SS- SSS SSS, ISN'II IS ITS: ‘Ifi, ‘ 111%“: III SSS‘S‘S SSS ' IS'S‘ ”SSS ‘ S S -‘ ‘é'l “SIMS "WIS S 5 I -,' 'd: » -:‘v ' . ”u— _ _ - _ ' ' - --'" . ’ ‘— c4... .. r—‘ .. - - - - fo- 2 . - _, I r? -.-. -- . m... "a” _.- _, I ,fl _I . . - - r.“ '- '5- ’ - - ' . . _I_ _ . ' O. 5 _ - ‘ I ‘ ' ’ A. ' .| ..-» .7. . . f. - ": 5' -’ It!) ”—- . -. -«u 4..- . _ .1—7. , _ .. ~ "0—! ‘ _-I.L—'-.r.‘__— .. 1 F. " ... fl _ .-4 C02 Val. a-KIV a-k-glutarate Glutamate Free NH3 lutamine alanine Pyr. <—— ‘— Glucose Figure l. The pathway of alanine and glutamine synthesis in muscle. 18 of alanine, while exogenous glucose or muscle glycogen provides the pyruvate (Chang and Goldberg, 1978a). Tishler and Goldberg (1980) indicated that under certain conditions, muscle may release even greater amounts of glutamine than alanine. Odessey gt 31. (1974), and Chang and Goldberg (1978b) believe that the origin of the carbon skeleton of glutamine are those AAs that are generated by muscle protein degradation and then enter the tricarboxylic acid cycle (i.e., valine, isoleucine, aspartate, asparagine and glutamate). When rat diaphragm is incubated jn_yitrg, it releases much lower amounts of these five amino acids than would be anticipated from the composition of muscle protein. The missing amounts of these AAs together equaled the amount of glutamine synthesized de novo by the muscle. These find- ings suggested conversion of the carbon skeletons of these AAs into glutamine. B. Alteration in AA Metabolsim in Skeletal Muscle In nonruminant as well as in ruminant, starvation, hormonal administration and altered protein intake can result in significant alterations in amino acid concentration in plasma (Clark et 11., 1968; Munro, 1979; Felig gt al., 1970; Adibi, 1971; Hambraeus et_al,, 1976; Bergman and Heitmann, 1978; Hutson and Harper, 1981; and Motil et al., 1981), as well as the arteriovenous concentration differences (Aoki gt .31., 1973; Bell et _l., 1975; Ballard _t._l., 1976; and Heitmann and bergman, 1978) which can be attributed to change in AA metabolism in the tissues. 1. Effect of dietary protein on AA metabolism in skeletal muscle Clearly, one would anticipate marked changes in amino acid metabolism 19 following the ingestion of a protein meal. Hutson and Harper (1981) showed that blood BCAA concentrations of rats fed high protein diet were elevated two- to three-fold. Branched-chain keto acid concentra- tions were also increased about two-fold. With respect to the effect of protein ingestion on muscle AA exchange, data are not available in ruminants. However, studies in the rat have demonstrated a net uptake by peripheral tissues of the BCAA in the ab- sorptive period (Yamamoto et al., 1974). Studies with normal humans reveal selective splanchnic release of BCAA and uptake by muscle tissue which persists for at least three hounsafter protein ingestion (Felig, 1975). In contrast, alanine output from muscle continued unchanged or is reduced for only one hour after protein intake (Aoki et al., 1973; Yamamoto t al., 1974; and Felig, 1975). Working with young men, Hambraeus _t _l. (1976) suggested that dietary leucine facilitate both tissue uptake of BCAA and their intra- cellular metabolism. Motil _tugl. (1981) fed a group of young men three different levels of protein (a surfeit level, a level approximating maintenance requirements, or a grossly inadequate level). The change in protein intake from a marginal to a surfeit level was associated with an in- creased leucine flux and incorporation of leucine into body protein. In the fed state, oxidation of leucine increased sharply and release of leucine from tissue protein diminished. When dietary protein intake was reduced from the requirement tolan inadequate level, leucine flux and body protein synthesis and protein breakdown were reduced, also a smaller reduction in leucine oxidation was noted. When AAs are infused intravenously rather than ingested, thus 20 bypassing the gut and liver, the importance of alanine in AA catabolism and nitrogen disposal is also readily apparent (Felig, 1975). In the rat, infusion of each of 20 AAs resulted in increases in the alanine content of a variety of tissues (liver, kidney and muscle) (Coulson and Hernandez, 1968). From these observations it is clear that alanine is the major L vehicle of a-amino nitrogen output from gut and muscle in the fed as well as the_fasted state. 2. Effect of starvation on AA metabolism in skeletal muscle. The metabolic response to starvation has been described as biphasic, the change in body fuel metabolism differing in the early and late stages of fasting (Felig, 1975). The initial response is directed at maintain- ing hepatic glucose output by increasing gluconeogenesis, whereas the late response is directed at maintaining body protein reserves by minimizing protein catabolism. Since StOVEd liver glycogen lS rapidly depleted in fasting (Felig, 1975), initially there is an augmented hepatic uptake of glucose precursors, notably alanine, to maintain hepatic glucose output. This increase in gluconeogenesis observed during the first three days of starvation is a consequence of augmented splanchnic fractional extraction of alanine as well as increased release of this amino acid from muscle (Odessey and Goldberg, 1972; Odessey ‘_t.al., 1974; Felig, 1975; Ballard et_al,, 1976; and Bergman and Heitmann, 1978) which in turn comes from the breakdown of BCAA in skeletal muscle, as reviewed earlier. During starvation, plasma level of the EAA in sheep increased significantly (Leibholz and Cook, 1967; Hogan et al., 1968; and Leibholz; 1970). They attributed this large increase in plasma free AA to tissue 21 breakdown and AA release during starvation. In nonruminants skeletal muscle rapidly degrades the BCAA, This tissue is probably the major site for the degradation of these amino acids in the organism (Odessey and Goldberg, 1972, 1979; Adibi, 1976; Chang and Goldberg, 1978a,b; and Goldberg and Chang, 1978). Starvation enhances the catabolism of these AAs in man (Aoki et al., 1973; Felig, 1975; Hambraeus, 1976; and Motil t 1., 1981), in dogs (Nissen and Haymond, 1981) and in rats (Buse ;t al., 1973; Chang and Goldberg, 1978 a,c; Goldberg and Chang, 1978; Odessey and Goldberg, 1979; Hutson et al., 1980; Goodman _thal., 1981; and Hutson and Harper, 1981). However, 1. (1976) and Bergman and Heitmann working with fasted sheep, Ballard gt (1978) concluded that the concentration of the BCAA either decreased or remained unchanged after fasting. The arteriovenous differences HOth bYBallard_t__l_. (1976) showed that, after fasting, there was no real alanine release, while Bergmm and Heitmann (1972) showed an overall release of AAs by the hindquarters especially of alanine and glutamine, which is similar to nonruminant species. However, unlike nonruminant, BCAA are not totally catabolized in ruminant muscle. Bergman and Pell (1982) concluded that the rise in blood leucine concentration in starved sheep occurred because net leucine production by peripheral tissues overcompensated. for the negligible leucine absorption by the portal- drained viscera. Also, there was little or no concomitant increase in leucine utilization in other tissues. 3. Effect of insulin on AA metabolism in skeletal muscle. Amino acid metabolism in skeletal muscle is controlled by hormones (Manchester, 1970; Young, 1970; Call et_al,, 1972; and McLaughlan, 1974). 22 Insulin is thought to be one of the major regulators of muscle protein metabolism. Insulin and growth hormone are both necessary for growth of mammals and are usually considered to have the greatest effect on protein synthesis in muscle (Trenkle, 1974). The effect of insulin on AA metabolism has received considerable attention with respect to stimu- lation of protein synthesis (Munro, 1964; Manchester, 1970; McLaughlan, 1974; Bergman and Heitmann, 1978; and Goldberg et al., 1980), transport of amino acids into cells (Grubb, 1976; Hutson _t._l., 1980; Young, 1980; Fehlmann et al,, 1981; and Horowitz and Pearson, 1981) and inhibition of protein breakdown (Mortimore and Mondon, 1970; Fulks et_gl,, 1975; Rannels gt al,, 1975; Li and Goldberg, 1976; Rannels gt_gl,, 1977; and Goldberg, 1979). The predominant effect of insulin is to increase synthesis of all proteins in about the same proportion as in normal animals (Trenkle, 1974). The level of insulin is probably the most important factor re- gulating protein balance in skeletal muscle (Cahill gt_al,, 1972). After food intake, the elevated plasma levels of this hormone promote a net uptake of AAs by muscle and their incorporation into protein, while upon fasting, the fall in insulin leads to a net release of AA from muscle (Cahill et_al,, 1972; Felig, 1975; and Ruderman, 1975). The increase in plasma insulin level after a meal (Fajans and Floyd, 1972) or increase with diets containing protein would stimulate the uptake of amino acids by muscle, thereby protecting them from catabolism by the liver and result in overall improved utilization of AAs for protein synthesis rather than the stimulation of AA uptake by insulin being a prerequisite for protein synthesis. 23 Effect of three days of starvation on catabolism of leucine and a-keto isocaproic acid in skeletal muscle have been examined using a perfused hindquarter preparation of rats (Hutson et_al,, 1980). There was net release into the perfusate of total leucine carbon by hindquarter from starved rats, and no change with fed controls. After 30 minutes insulin addition resulted in net uptake of leucine from a reduced accum- ulation of a-keto isocaproic in the perfusate. With fed rat hindquarter, insulin addition resulted in a slower constant rate of oxidative decar- boxylation. In both fasted and fed groups leucine incorporation increased with insulin addition. A little earlier, Grubb (l976),working with the same perfusate,found that addition of insulin resulted in a significant increase in the rate of glucose uptake and de novo alanine production. Effect of insulin on AA metabolism has been also investigated with ruminants. Call et;_l, (1972) working with sheep, indicated that plasma free amino-nitrogen was slightly lower after insulin injection. The NEAA were reduced to 83% of the initial level and EAA to 66% of the initial level. Isoleucine, leucine, tyrosine, lysine, histidine, proline and arginine were significantly depressed. Apparent depression of alanine, valine, methionine and phenylalanine by insulin was less conclusive. Aspartic acid, threonine, serine and glycine were not influenced by insulin. t al. (1975) studied Brockman and Bergman (1975) and Brockman the effects of insulin on sheep. Insulin had no effect on net hepatic removal (Jr concentrations of the above amino acids. It did, however, decrease the concentrations of the BCAA indicating increased protein synthesis in muscle. 24 According to the review of Munro (1970) AA levels in the plasma can influence secretion of insulin. An oral dose of leucine causes hypoglycemia due to release of insulin. After a meal containing protein, the three BCAA often show the largest increments in the peripheral blood, which may have relevance for the increase in blood insulin level found after meals of protein. He also reported that insulin secretion is much more enhanced by a meal containing carbohydrate and protein than by giving carbohydrate or protein alone. In ruminant, however, Forbes (1980) reported that propionate plays the main role in stimulating insulin secretion. Bhattacharya and Alulu (1975) injected salts of the three major volatile fatty acids intraruminally in sheep and found that, although subsequent food intake was depressed by all treatments, only propionate significantly stimulated the secretion of insulin as measured in both portal and jugular blood. Insulin has been shown to increase transport of AAs into skeletal muscle cells (Snipes, 1967) as well as in freshly isolated rat hepa- tocytes (LeCam and Freychet, 1978; Fehlmann et al., 1979; and Fehlmann gt al., 1981). Membrane active transport was initiated by insulin (Horowitz and Pearson, 1981). Goldstein and Reddy (1970) found no stimulatory effect from insulin when muscle tissue was incubated in high concentrations of AAs and sug- gested that insulin exerts its effects on protein synthesis in muscle almost entirely on amino acid transport. Binding of insulin to specific receptors on the plasma membrane of target cells is important because of its role in mediation of hormone action (Etherton, 1982). Although insulin binding is essential for hormone action, binding may not represent the rate-limiting step in 25 the action of insulin. In his review, Trenkle (1974) reported that all muscle proteins continue to be synthesized, but in reduced amounts, in the absence of insulin. Also, this hormone is not always essential for an increase in protein synthesis to occur. Because in its absence, there is a signifi- cant increase in protein content of muscle during work-induced growth (Goldberg 1967; 1968). MATERIALS AND METHODS I. Experiment One A General Design Eight crossbred steers with an average body weight (BW) of 333 kg were fed a 9.5 % crude protein (CP) semipurified ration (Table 1) once daily at 9 a.m., at 2% (6.7 kg) of their BW. The steers were housed individually in 2 x 2 m metal metabolism stalls with free access to water. Prior to the start of the experimental period, the steers were adapted to the diet for a 21-day period. In the experimental period the steers were divided into two groups of four steers each. Each group was assigned to a split-plot design (animals as incomplete blocks) Gi11,(1978; 1980) (Table 2). The amino acids utilized were thsomers of methionine and cysteine obtained from Sigma Chemical Company. In this study, the steers were injected intraperitoneally (IP) twice daily at 9:00 a.m. and 9:00 p.m. for five days with graded levels of methionine and methionine plus cysteine; the amounts of amino acids injected during each treatment period are presented in Table 2. Methionine was diluted in sterile saline solution in a proportion of 1:20 (w/v) and cysteine was diluted in a ratio of 1:10 (w/v). The pH of the solutions were raised to 6.5 using 6 N NaOH to prevent damage to the peritoneum. The steers were given a five-day rest period after each treatment period to eliminate carryover effects from the previous treatment. Blood samples were collected from the right jugular vein of each 26 27 TABLE 1. Composition of the diet used in Experiments One and Two 22:22:32? . Oats, grain (4) 4-03-309 10.00 Wheat, bran (4) 4-05-191 5.00 Corn, dent yellow grain grnd 2 US mm wt 54 (4) 4-02-931 51.55 Soybean seeds, solv-ext, grnd ms 7% fiber (5) 5-04-604 3.75 Corn, Cobs, grnd (1) l-02-782 20.00 Sugarcane, molasses, mm 48% invert sugar mm 79.5 degrees brix (4) 4-04-696 5.00 Wheat, flour byproduct, fine sifted ms 4% fiber (4) 4-05-203 1.00 Urea (45% N) 0.25 Limestone, grnd, mn 33% calciuma (6) 6-02-632 1.45 Trace mineral and vitamin mix 2.00 Crude Protein (N x 6.25) 9.50 aCalcium Carbonate. b Contained in %: Zn, mn 0.35; Mn, mn 0.2: Fe, mn 0.2: mg, mn .15: Cu, mn 0.03; Co, mn 0.05; I , mn 0.007; NaCl, mx 98.5, Vitamin A palmitate, 2,000,000 IU/tonzof final diet; Vitamin 0, 250,000 IU/ ton; Vitamin E, 55,000 IU/ton. 28 TABLE 2. Experimental Design of Experiment One Steer Number Period 1 ~ 2 3 4 l A H B E 2 B D E C 3 F B G A 4 G E A D 5 C F H G 6 H C D F Treatments AA injection level (Q/d) Group I Group II Met. Cyst. Met. Cvst. Code 0 0 0 7 H 2 0 2 7 G 4 o 4 7 ' c 6 0 6 7 D 8 0 8 7 B 10 0 10 7 A 12 0 12 7 F 14 0 l4 7 E 29 steer at the fifth day of the treatment period, two hours after the morning injection. This experiment took place between August and December 1979, the steers showed an average daily weight gain of .34 kg during that period. Sample Processing Approximately 12 ml. of blood were collected in heparinized vacu- tainer tubes. Plasma was then obtained by centrifugation and 2 ml were deproteinized and prepared for AA analysis according to the procedures described by Bergen et al. (1973) and then frozen at -10° C until analysis. The remaining plasma was also frozen at ~10° C for the determination of plasma urea nitrogen (PUN). Chemical Analyses A. Plasma methionine Methionine concentration was determined from the protein free filtrate by means of Ion Exchange Chromatography (with a Durrum Chroma- t __1_. (1973). tography Amino Acid Analyzer Kit) according to Bergen 8. Plasma urea nitrogen (PUN) Plasma urea nitrogen levels were determined by Conway Microdif- fusion method (1960) as outlined by Fenderson (1972). Statistical Analysis The data obtained were statistically analyzed according to Gill (1978). 30 II. Experiment One 8 General Design This study was carried out at the dairy farm between September and November 1980. Six male Holstein calves with an average body weight of 88 kg and approximately two months of age were used in this experiment. These animals were weaned and their rumens were developed, but kept with their active esophageal groove as means of rumen bypass by feeding milk once a day for at least two weeks before the experiment started. The calves were divided into two groups of three each, in a com- pletely randomized design (Gill, 1978). They were housed individually in 1.5 x 1.8 m metal stalls with free access to water. Due to disease one calf of the group fed milk replacer supplemented with lysine died during the last week of the experimental period, there- fore, the data for the last week is an average of two observations. Milk replacer (Table 3) supplied by Milk Specialties Co., Dundee, Illinois, were fed at 60% of the DM requirements (NRC, 1978). (To complete the DM requirement the ration shown in Table 1 was weighed and offered twice daily at 7:00 a.m. and 4:00 p.m. Feed refusals, if any, were collected and weighed daily before the next feeding for DM intake measurement. Milk replacers were diluted to 13% solids with water (370 C), mixed with a hand beater, and fed to the animals by nipple pail in two equal meals at 7:00 a.m. and 4:00 p.m. All animals were weighed at biweekly intervals and the amount of feed intake were adjusted according to body weight changes to calculate 31 TABLE 3. The composition of milk replacers used in Experiment Two. Milk replacer Iggredients __1L__ __j;__ Dried skim milk 50 50 Protein-fat mix (12/50) 20 20 Corn gluten meal 20 20 Dextrose 8 8 Premix of vit. and minerals 2 2 Lysine* - + * Lysine was added to milk replacer B at level of .7% on the DM basis. the G/F ratio. At weeks 2, 5, and 8 blood samples (approximately 12 ml) were col- lected immediately before morning feeding and then at l, 2, 4, and 6 hrs after feeding. All samples were processed as described in experi— ment one. Plasma lysine determination Plasma lysine concentration was determined from the protein free filtrate by means of Ion Exchange chromatography (with a Durrum Chroma- tography Amino Acid Analyzer Kit) according to Bergen gt_ 1. (1973). Statistical Analysis The data obtained were statistically analyzed according to Gill (1978). III. Experiment Two General Design Eight crossbred steers were used to study the effect of dietary protein on the arteriovenous concentration difference. Animals were approximately 15 months old with an average BW of 510 kg. All animals were accustomed to metabolism cages and to frequent handling. Prior to the start of the experimental period, the steers were adapted for two weeks to the control diet No. 1 (Table 4). They were then divided into two groups of four steers each. Each group was fed either high or low protein diet (Table 4) for at least two weeks before sampling. Feed intake was restricted to 90% of the gg lib. to insure cleanup. All animals had free access to water. 33 TABLE 4. Composition of diets used in the Third Experiment Ingredient International Diet reference no. 1 2 3 ----- % of 0M - - - Corn, aerial pt, W. ears ensiled, mature, well- eared, mx 50% mn, 30% dry matter 3-08-153 72.5 72.5 72.5 Corn, dent, yellow grain, gr 2 US (4) 4-02-931 20.0 24.0 12.0 Soybean, seeds, Solv-ext. grnd, ms 7% fiber (5) 5-04-604 4.0 - 12.0 Limestone, grnd, a mn 33% calcium (6) 6-02-632 1.5 1.5 1.5 Trace mineral and vitamin mix 2.0 2.0 2.0 Analysis Crude protein (%) 12.8 9.4 19.74 Total Digestible Nutrient (%) 79.0 79.4 77.50 Dry Matter (%)' 43.81 42.91 42.91 aCalcium Carbonate. b ton; Vitamin E, 55,000 IU/ton. Contained in %: An, mn 0.35; Mn, mn 0.2; Fe, mn 0.2; Mg, mn 0.15; Cu, mn 0.03; Co, mn 0.05; 12, mn 0.007; NaCl, mn 98.5, Vitamin A palmitate, 2,000,000 IU/ton of final diet; Vitamin 0, 250,000 IU/ 34 Five days before sampling, steers under went surgical procedures. Surgical Procedures At least five days before sampling the steers were placed under general anesthesia for surgical implantation of the saphenous artery and vein cannulas. 0n the inside of the thigh an incision was made over the saphenous artery and vein. The two vessels were exposed and a polyvinyl tube (1.5 mm 10, 2.32 mm 00) was inserted into each vessel and passed under the skin for a distance of 30 cm to the outside of the thigh. These cannulas thus gave an arterial and venous blood sample draining the hind limb. A11 cannulas were filled with sterile saline containing 100 U of Heparin/m1 and were flushed daily. Both sides were cannulated, so if one side was clotted the other side could be patent. Blood Sampling and Processing All animals fed control, high protein or low protein diets were sampled immediately before the morning feeding and then 2, 4 hours after feeding. The insulin treated animals were sampled right before the injection and then 1, 2 and 4 hoursafter injection. The starved animals were sampled once after 24 hour starvation, the second samp- ling was taken after 48 hour starvation. Twenty ml blood were taken from each of the two vessels at the same time and transferred to heparinized tubes and placed in ice. The plasma was obtained and prepared for AA analysis as described in Experiment 1. After the blood sampling, only two steers were kept with their cannulas open, and they were used as control for this group. They 35 went through the same feeding procedure but on the control diet. Another group of four crossbred steers were used through the same procedure to study the effect of insulin injection and starvation on the arteriovenous concentration difference. They were fed the control diet No. 1 (Table 4) for two weeks, then went under the cannulation and sampling procedures as control for this study. Two days later all four steers were injected with insulin (.20 units/kg BW) and the blood sampl- ing repeated. The animals were then starved for 24 and 48 hours and the sampling procedure repeated. Plasma Flow Measurement A. Blood Samples Para-amino hippuric acid (PAH) was infused into the jugular vein as a blood flow marker (Katz and Bergman, 1969). The PAH solution used was 10% (w/v); a primer dose of 15 ml was administered and was followed by continuous infusion at 45.8 mg/hr for three hours. Blood samples (5 ml each) were taken simultaneously from both cannulas at 100, 120, 140, 160 and 180 min. after the start of the PAH infusion. The blood was transferred to heparinized tubes and placed in ice. Immediatedly after the collection of blood samples, packed cell volume (PCV) was determined by centrifugation of capillary tubes con- taining whole blood. The remainder of the sample was used for PAH determination. B. Para Amino Hippuric Acid Determination Para amino hippuric acid concentration in blood samples was de— termined by procedures described by Smith gt_gl, (1945) and modified 36 by Katz and Bergman (1969). (Appendix A). C. Calculations I F = B Cv - CA _ 100 - PCV Fp ' FB x 100 Net uptake (u mole/hr) = Fp (CXA - va) where: FB = Blood flow (ml/min) H II Infusion rate of PAH (mg/min) Cv and CA = The concentration of PAH (mg/ml) in the venous and arterial blood respectively. Fp = Plasma flow (ml/min) PCV = Packed cell volume (%) va and CxA = AA concentration in venous and arterial plasma (n moles/ ml) respectively. Statistical Analysis The data were statistically analyzed according to Gill, (1978). RESULTS AMINO ACID REQUIREMENT STUDIES EXPERIMENT ONE A PlaSma methionine response to IP injections of methionine and methionine plus cysteine as criterion of methionine requirement: The purpose of this study was to measure plasma methionine response to incremental levels of IP injection of methionine and methionine plus cystehuain order to evaluate the methionine requirement in growing steers in a manner similar to Fenderson and Bergen (1975) and Towns and Bergen (1979). Plasma methionine was expected to remain at a low, relatively constant level until the requirement was reached, after which it increased rapidly with higher injection level. The injection level, at which the plasma methionine concentrations begin to deflect upward, is considered the requirement. The effect of incremental levels of methionine on its respective plasma concentration is presented in Table 5. Plasma methionine started to increase at the injection level of 4 g/d with a linear but low rate, and started to accumulate at the injection level of 8 g/d with a linear and higher rate after each successive increment. Since the same steers fed the same diet throughout the study were used, pre- injection plasma levels from the first day of the experiment were con- sidered to represent the basal concentration of plasma methionine. The point at which a line representing this basal level was intersected by a regression line obtained from the incremental levels of plasma 37 38 TABLE 5. Plasma Methionine and Plasma Urea-N Responses to IP Injection of Graded Levels of Methionine Met. Injection ' Plasma Methignine Plasma Ugea-N Level u mole/d1 mg/dl 0 1.25 i 0.26 7.84 _+_ 0.55 2 1.57 :_0.12 NS 7.39 :_0.75 NS 4 2.20 :_0.06 * 8.45 :_0.31 NS 6 2.52 :_0.16 * 7.20 :_0.51 NS 8 3.36 i_0.31 ** 7.36 i 0.61 NS 10 5.06 :_0.36 ** 7.28 i 0.81 NS 12 7.24 :_0.55 ** 7.84 :_0.55 NS 14 10.75 :_2.13 * 8.07 :_0.71 NS aGrams per day per steer. bMean and standard error of four steers. *Statistically significant (from the zero level) at the 5% level. **Statistically significant (from the zero level) at the 1% level. NSNon-significance. 39 methionine is the so called breakpoint at which plasma methionine would start to accumulate above the basal line with each increase in the IP methionine injections and was therefore considered to represent the requirement. This breakpoint was equivalent to an IP injection level of 6.8 g of methionine per day (Figure 2). Incremental levels of methionine along with 7 g of cysteine per day were injected intraperitoneally to determine if cysteine can supply part of the total sulfur amino acid need and can thus spare methionine. Under the conditions of this study plasma methionine increased immediatly (Table 6) with the first injection level (7 g cysteine + 0 g methionine) and then continued to increase rapidly with each successive increment. The biological interpretation of these data (Bergen, 1979) is that meth- ionine could no longer be shown as limiting in the presence of adequate cysteine and that methionine passing to the small intestine from the rumen-reticulum satisfied the requirement of this AA and the cysteine and methionine combined met the TSAA requirement. The plasma methionine response to incremental IP injection of cysteine plus methionine is presented in Figure 3. Plasma urea nitrogen concentration (Tables 5, 6) were not in- fluenced by either of IP methionine or methionine plus cysteine. Plasma urea nitrogen levels remained consistantly low through the experimental period with values ranging from 6.72 to 8.68 mg/dl. This was expected because the highest injection level of 21 g/d (14 g methionine plus 7 g cysteine) which represents only 3.3% of the total N intake and administered postruminally would not be expected to affect the plasma urea nitrogen substantially. Plasma methionine (u mole/d1) 12 11 10 40 f ’ ' Breakpoint at 6.8 g/d A l j I l 1 ___A 2 4 6 8 10 12 14 Methionine injection (g/d) Figure 2. Plasma methionine response to IP injection of graded levels of methionine ** Correlation statistically significant (P<.Ol). 41 TABLE 6. Plasma Methionine and Plasma Urea-N Responses to IP Injection of Graded Level of Methionine Plus a Constant Level of Cysteine. M aTreatment a Plasma Methignine Plasma Ugea-N et. Cyst. u mole/d1 mg/dl 0 7 2.36 :_0.81 8.68 :_0.27 2 7 3.46 :_1.03 NS 6.91 :_0.99 NS 4 7 7.29 :_1.06 ** 7.54 :_0.26 NS 6 7 7.47 :_l.34 ** 7.47 :_0.37 NS 8 7 5.56 i 1.02 * 7.65 :_1.53 NS 10 7 10.55 :_3.26 * 8.40 :_0.55 NS 12 7 12.26 :_2.43 ** 7.47 :_0.49 NS 14 7 9.44 :_2.42 * 6.72 + 1.17 NS aGrams per day per steer. bMean and standard error of four steers. *Statistically significant (from the zero level) at the 5% level. **Statistically significant (from the zero level) at the 1% level. NSNon-significance. Plasma methionine (u mole/d1) 14 13 12 11 10 42 Methionine Y = 0.6x + 3.1 ¢ * i Y' = 0.86 /. ' I. l. 2 4 6 8 10 12 14 Methionine injection (g/day) Figure 3. Plasma methionine response to IP injections of graded levels of methionine supplemented with cysteine. * Correlation statistically significant (P<.05). 43 EXPERIMENT ONE 8 This experiment was conducted to examine the use of the esophageal groove as a method of rumen bypass to study amino acid requirements of young ruminant with developed rumen. The calves used in this study were weaned and fed dry feed to inSure rumen development. Lysine addi- tion to a gluten-based milk replacer fed at 60% of the required dry matter (NRC 1978) was used as an experimental variable. Animal perfor- mance and plasma lysine concentration were used as indicators of the occurence of rumen by-pass. Table 7 shows changes in body weight, average daily gain (ADG), dry matter (DM) intake and gain/feed (G/F) ratio of the animals. All animals gained weight during the experiment. However, the lysine-supplemented group gained significantly more weight (.84 kg/d) (P<.05) than the unsupplemented group (.63 kg/d). Daily gain was within the range of gain (.36 to .90 kg/d) reported by NRC (1978). It was also observed (as shown in Table 7) that the DM intake was not significantly different between the two dietary regimes. The data in the same table indicates that the G/F ratio was signifi- cantly (P<.05) increased from 0.2 to 0.29 when lysine was added to the milk replacer. Plasma lysine concentration at different times after feeding are summarized in Table 8. In both groups plasma lysine concentration sig- nificantly (P<.05) increased after feeding, reaching the maximum level at 2 hours after feeding (Table 8 and Figure 4). However, at all times after feeding plasma lysine concentrations were significantly (P<.05) ‘44 TABLE 7. Effect of Feeding Milk Replacers Supplemented with or without Lysine 0n Calf Performancea Parameter Milk Replacer with lysine without lysine No. of animals Initial BW (kg) Final BW (kg) Duration of expt. (weeks) ADG (kg/d) 0M intake (kg/d) G/F ratio 3 3 87.1 : 9.3b 88.6 i 9.1 NS 138.3 _+_ 15.5 119.5 _+_ 10.7 * 8 8 .84 i .06 .53 i .03 * 3.39 i .29 3.38: .19 115 .29 _+_ .014 .20 1.03 * aCalves were weaned and fed dry feed to start rumen development and the esophageal groove reflex was kept operative by feeding milk once daily. bMeans 3; SE. Milk replacer constituted 60% of daily dry matter intake. * Statistically significant at the 5% level. NSNon-significance. 45 TABLE 8. Plasma Lysine Response to Time after Feeding Milk Replacer Supplemented with or without Lysinea 1 - Milk Replacer Parameter with lysine without lysine u mole/d1 1. hrs after feeding 0 1.81 i 0.21” 1.08 2‘. 0.17” 1 8.76 _+_1.50”’e 4.05 351.07” 2 11.99 i 1.27” 4.09 i: 0.76d 4 11.431195” 1.50_+_0.15” 6 6.53:1.11e 1.73:0.29” II. weeks of feeding 2 2.72:0.46b 1.99:0.39” _ 5 7.76 i 1.88” 3.18 1 0.44” 8 10.27 _+_ 2.33” 4.93 i 2.13” aValues are means and standard error of three calves. b’c’d’eValues not sharing common superscript in rows or columns were significantly different (P<.05). Plasma lysine (u mole/d1) 46 _+ lysine ""'""- lysine ,I”’ ~~‘\ I [/1 \\ / \\ 12. ’ \ .. / \ / \ n. / ‘-_‘ \ I fi' ‘\\ \ 10J 7 7’ ‘\ \\ I ’ \ \ 91 I I \ \ I \ \ II I \\ \ I \ 84 l I \\ \ . I I \ 7. I , \ I I \ l ’ ' \\ 5 l 1’ \x ’ I -.. 5' I] -‘ I] l’ ’- \\ 44 ll] ’ \ \\ l I ‘5 \ 3‘ I” l’ ’ \\\\ \ I I ’l \ \ I’ ll \ \ I’/ I \ \ 2| I’I/ \ \\ I \\\\~ __________ T 1 ~ ~l-:::::: :-.:-::- 0 l 2 4 6 hrs after feeding Figure 4. Plasma lysine response to time after feeding of young ruminant calves fed milk replacer (at 60% of DM intake) supplemented with or without lysine. 47 higher for the lysine-supplemented group than lysine level in plasma of the calves fed the replacer devoid of supplemented lysine, The plasma lysine in the latter group decreased to prefeeding_ levels four hours post feeding, while in the lysine-supplemented group plasma lysine con- centrations did not return to prefeeding levels even after six hours (Figure 4). Table 8 also shows that in both groups, the plasma lysine concentrations, sampled at two hours after feeding increased throughout the experimental period. In the lysine-supplemented group plasma lysine increased atasignificantly (P<.05) higher rate than the unsupplemented group, which also increased in linear pattern. This indicates that the lysine intakes for both feeding regimes were apparently exceeding the lysine requirements especially at five and eight weeks of the experiment. DISCUSSION AMINO ACID REQUIREMENT STUDIES EXPERMENT ONE A The plotting of a two-phase plasma amino acid response curves is a well established procedure in the study of AA requirements (Zimmerman and Scott, 1965; Mitchell §t_al,, l968; Stockland gt_gl,, l970; Young _t__l_., l97l; Brookes e_t_fl., 1973; Reis 9131., l973; Broderick §_i;_a_l_., l974; Tao _t_gl,, l974; Fenderson and Bergen, 1975; Williams and Smith, l975; Foldager gt 31,, l977; Towns and Bergen, l979; Tzeng and Davis, 1980). Plasma met responses with IP supplementation of met alone (Figure 1,2) are in agreement with the results of Fenderson and Bergen (1975) and Towns and Bergen (1979); who, working with growing steers fed a similar ration, found that the methionine supply in abomasal flow was limiting. Methionine was also the first limiting AA in the studies of Nimrick et_al, (1970a) for growing lambs, Wakeling t 31, (1970) for sheep, Richardson and Hatfield (l978) for growing steers and Tzeng and Davis (1980) for young calves. The methionine needed above that SUpplied from digesta flow reported in the literature vary from 2 to l4 g/d. Williams and Hewitt (1979) reported a methionine requirement of the preruminant calf of 2.1 g/d (depended on the lysine/methionine ratio in the carcass), while Williams and Smith (1975) and Towns and Bergen (l979) found the supplemental re- quirement to range between 3.8 and 4.5 g/d. The methionine requirement 48 49 t al. (1977) for preruminant calf and the supple- reported by Foldager mental methionine reported by Fenderson and Bergen (1975) for growing steers ranged from 5.9 to 7 g/d. The highest methionine requirement reported for the young calf was by Tzeng and Davis (1980) at 10.2 to 13.8 g/d. Using the two different accumulation ratios (in the manner of Tzeng and Davis, 1980) the two regression lines (Figure 2) intersected at a breakpoint equivalent to an IP injection level of 6.8 g/d. Therefore, the supplemental methionine requirement over the digesta flow is 6.8 g/d. This value is in agreement with those reported by Fenderson and Bergen (1975) and Foldager _tual. (1977). The immediate and linear increase in plasma methionine during IP injection of cysbyhe plus methionine indicated that methionine was spared in the presence of adequate (or probably excess) cysteine. Methionine also was not limiting in the study of Hill _t_gl, (1980) when infused postruminally to growing steers fed a urea-supplemented diet adequate in sulfur. This response was similar to that in lambs insted with 3 g methionine or higher daily (Strath and Shelford, 1978). How- ever, the present data disagree with those of Towns and Bergen (1979); who, working with growing steers fed a similar diet, obtained a break- point on the plasma methionine response curve at 3.8 g/d when methionine was injected with 7 g cysteine. This.disagreement probably was because their last treatment did not produce a linear increase in plasma meth- ionine response curve, but a smaller increment which resulted in a 1 sigmoid-type response. Therefore, not to destroy the linearity of the plasma response curve, these workers avoided taking this point into account. Also, plasma methionine concentratiors at the injection 50 levels of 0 and 3 g/dwere the same; the overall linear regression does not apply. This type signoid shape was reported by others (Mitchell gt 51. 1968; Young _e_t_a_]_. 1971). In ruminants, the determination of AA requirements must include an estimation of the abomasal flow of Ms so that the total (abomasal flow plus the injected amount) intake can be properly evaluated (Fenderson and Bergen, 1975). The methionine requirement was calculated by adding the amount of injected methionine reeded to produce the breakpoint in the plasma response to the amount of methionine absorbed across the intestinal epithelieum. The amount of dietary N and AA reaching the abomasum in steers in this study appears in Table 9. The data derived in this experiment was based on the passage studies with the same diet as reported by Fenderson and Bergen (1975). Using the DM intake of 6.7 kg/d of the present study, dietary N reaching the abomasum was 99.82 g/d which re- presents 98% of the 101.84 9 of N ingested daily. Amino acids reaching the abomasum amounted to 5.03 g/d for cystine, 10.72 g/d for methionine and 15.75 g/d for TSAA. Table 10 contains the quantitation of the methionine requirement. Hogan (1973) determined that the digestibility of bulk protein in the small intestine of ruminants was 70%. In the present calculation it was therefore assumed that 70% of the protein reaching the abomasum and small intestine would be digested and absorbed. Hence, the absorbed values were 7.5 g/d of methionine and 3.52 g/d of cystine, adding up to 11.02 g/d of total sulfur amino acids. Based on the data obtained here with those of Fenderson and Bergen (1975), the absorbable methionine requirement ranges from 7.5 to 14.3 g/d, while that of TSAA (absorbable) was 17.82 g/d. This is in 51 TABLE 9. Nitrogen and Amino Acid Passage in Steers Fed the 9.5% Crude Protein Ration Utilized in Experiment One.a Item g/kg feed g/dayb Nitrogen 14.9 99.83 Crude Protein” 93.1 623.77 Methionine 1.6 10.72 Cystine .75 5.03 TSAAd 2.35 15.75 aBased on data by Fenderson and Bergen (1975). ”Dry matter intake 5.7 kg/day. cN x 6.25. dTotal sulfur amino acids. .mkum ocwsm Lampzm Pmuohu Ammmpv :moo: a Amumpv :wmemm use cemcmucmu an mumc :o ummmmm 2 5’ Nw.np Nw.u_ o.“ m.o No.PF om mm.mp <WDCD F12 > @5357 m2 >W€D mmz 3797:“; E” 33' ‘"2'5" im 37°? '53:?” > E E E i E Figure 5. The net amino acid uptake (or release) by the hind limb of steers fed the control diet (I). 63 TABLE 15. The Arteriovenous Concentration Difference and the Net Uptake of Amino Acids by the Hind Limb of Steers Fed the Low Protein Diet (Immediately Before Feeding) Plasma AA level (p mole/d1) Net uptake AA Arterial Venous Difference m mole/hr Asp 1.96 1. .23 2.24 1 1.29 - .28 1_ .06 - .73 1_ .16 Thr. 6.01 1_ .24 6.54 1. .66 - .53 1_ .36 -l.37 1_ .93 Ser 15.65 1 3.13 14.45 1 1.76 1.20 1 1.87 3.11 1 3.55 Asn 1.40 1_ .15 1.83 1_ .16 - .43 1_ .05 -l.11 1_ .13 Glu 11.81 1_ .53 11.47 1 1.38 .33 1. .85 .85 1 2.20 Gln 14.99 1 1.18 18.18 1 1.76 -3.19 1_ .53 -8.26 1_1.37 Gly 22.79 1 3.56 22.81 1 3.80 -0.02 1_ 24 - .05 1_ .62 Ala 12.21 1 1.74 26.05 1 1.56 -13.84 1_ 78 39.95 1_ .47 Pro. 9.27 1 1.01 9.12 1_ .42 .15 1_ .58 .39 1 1.53 Val. 16.12 1 1.63 16.22 1 1.43 - .10 1_ .26 - .26 1_ .52 Cyst 1.21 1_ .09 1.37 1_ .08 - .16 1_ .07 - .41 1. .03 Met, .73 1_ .11 .35 1_ .14 .38 1_ .03 .98 1_ .08 Ile 7.14 1_ .55 7.45 1_ 82 - .31 1_ .27 - .80 1_ .70 Leu 10.56 1_ .34 11.47 1 1.91 - .91 1_ .67 -2.36 1 1.74 Tyr 2.81 1_ .10 3.40 1 1.45 - .59 1_ .35 -1.53 1_ .91 Phe 4.05 1_ .14 4.61 1 1.42 - .56 1. .28 -1.45 1_ .73 Orn 11.45 1_ .69 11.79 1 1.32 - .35 1_ .63 - .90 1_1.63 Lys 7.63 1_ .46 7.86 1_ .83 - .23 1, .42 - .60 1_l.09 His. .18 1_ .67 6.08 1. .28 .10 1_ .39 .26 1 1.01. Arg. 5.23 1. .90 6.33 1. 99 -1.10 1. .69 -2.85 1_ .23 EAAa 63.65 1 5.04 66.91 1_6.57 -3.26 1 1.58 -8.44 1_3.96 NEAAb 105.55 112.41 122.71 112.86 -17.16 1 3.45 44.44 1 1.66 BCAAC 33.82 1 2.52 35.14 1 8.26 -1.32 1_ .74 -3.42 1_1.92 Values are means and standard error of four steers. The (-) indicates the net release of AA. aEAA = Essential amino acids. b NEAA = Non-essential amino acids. cBCAA = Branched-chain amino acids. 64 TABLE 16. The Arteriovenous Concentration Difference and the Net Uptake of Amino Acids By the Hind Limb of Steers Fed the Low Protein Diet (Two Hours After Feeding) Plasma AA level (n mole/d1) Net uptake AA Arterial Venous Difference m mole/hr Asp 2.74 1 1.05 1.94 1_ .85 .80 1_ .7 2.07 1 1.81 Thr 6.59 1_ .50 5.55 1_ .69 1.04 1_ .79 2.69 1_ .49 Ser. 19.17 1_1.30 10.29 1 1.63 8.88 1. .27 23.00 1_ .70 Asn 1.64 1_ .05 1.71 1_ .25 - .07 1_ .25 - .18 1_ .57 Glu 12.46 1 1.85 9.82 1 1.66 2.64-1. .79 6.84 1_2.05 Gln 14.36 1_ .48 25.85 1 3.99 -11.49 1 2.61 -29.26 1_6.76 Gly 24.61 1 2.16 21.03 1_1.76 3.58 1 1.40 9.27 1 1.04 Ala 11.48 1 1.66 26.13 1 1.44 -l4.65 1 1.22 -37.94 1. .57 Pro 9.47 1_ .99 6.45 1 1.41 3.02 1_ .58 7.82 1 1.50** Val 16.60 1 1.27 15.08 1_1.06 1.52 1_ .27 3.94 1_ .70 Cyst 1.34 1_ .09 1.05 1_ .68 .29 1. .01 .75 1_ .03** Met 1.28 1. .74 .53 1_ .22 .75 1_ .32 1.95 1 1.35 Ile 8.54 1_1.08 6.85 1 1.61 1.69 1_ .47 4.38 1 1.22 Leu. 12.86 1 1.99 10.60 1_2.56 2.26 1_1.43 5.85 1 3.70 Tyr 3.72 1_ .58 3.04 1_ .88 .,68 1_ .20 1.76 1_ .52 Phe 5.53 1_ .65 4.02 1 1.30 1.51 1_ .35 3.91 1_ .91 Orn 16.52 1_ .05 10.46 1 2.17 6.06 1 1.88 15.69 1 4.87 Lys. 11.01 1_ .03 6.97 1 1.78 4.04 1 1.25 10.46 1_3.30 His. 6.31 1_ .66 4.30 1 1.27 2.01 1_ .39 5.21 1_1.01** Arg. 6.70 1_ .81 6.35 1 1.49 - .35 1_ .32 .91 1_ .83 EAAa 75.42 1 9.73 60.25 1 7.92 15.17 1 4.81 39.29 112.46' NEAAb 117.51 113.24 117.77 111.62 -0.26 1 2.22 0.67 1 5.75** BCAAC 38.00 1 4.34 32.53 1 2.77 5.47 1 2.17 14.17 1 5.62 Values are means and standard The (-) indicates the net release of AA. aEAA = Essential amino acids. ”NEAA ”BCAA ** H I Significantly different than the control group (P<.Ol). error of four steers. Non-essential amino acids. Branched-chain amino acids. 65 TABLE 17. The Arteriovenous Concentration Differences and the Net Uptake of Amino Acids by the Hind Limb of Steers Fed the Low Protein Diet (Four Hours After Feeding). Plasma AA level (u mole/d1) Net uptake AA Arterial Venous Difference m mole/hr Asp. 1.81 1_ .17 1.45 1_ .16 .36 1_ .07 .93 1_ 19* Thr 6.24 1_ .36 5.77 1_ .63 .47 1_ .26 1.22 1_ .67 Ser 18.97 _ 1.40* 8.14 1_1.64 10.83 1_ .36 28.05 1_ 93* Asn 1.78 1 0.15 1.96 1_ .67 - .18 1_ 08 - .47 1_ 21* Glu. 9.23 1 2.52 8.96 1_1.13 .27 1_ .39 .70 1 3.60** Gln 16.88 1 1.91 28.13 1_2.46 -11.25 1 1.45 —29.14 1 3.76 Gly 28.87 1_ .56 21.75 1 1.66 7.12 1_ .50 18.44 1 1.30 Ala. .96 1 1.20 26.55 1 1.78 -17.59 1_ .53 -45.56 1 1.37* Pro 9.62 1_ .65* 7.68 1_ .71 1.94 1_ .66 5.01 1_ .16** Val 18.17 1_ .75 15.85 1 3.47 2.32 1 2.72 6.01 1_7.04 Cyst 1.34 1. .13 1.20 1_ .17 .14 1_ .02 .36 1_ .05* Met .61 1_ .31 .55 1_ .18 .06 1_ .18 .16 1_ .34 Ile. 7.82 1_ .46 6.60 1 1.53 1.22 1_ .67 3.16 1. .18 Leu. 11.37 1_ .53 10.42 1 2.68 .95 1_ .15 2.46 1_ 39 Tyr 3.17 1_ .21 2.57 1_ .88 .60 1_ .17 1.55 1_ .44 Phe. .69 1_ .26 3.96 1_ .91 .73 1_ .15 1.89 1_ .39 Orn 12.54 1_ .75 9.36 1_ .87 3.18 1_ .12 8.24 1_ .32 Lys .36 1_ .50 6.24 1_ .58 2.12 1_ .68 5.49 1. .21 His .41 1_ .43* 5.12 1_ .47 1.29 1_ .64 3.34 1_ .10** Arg. 7.53 1_ .41 5.65 1. .36 1.88 1. .05 4.87 1_ .13 EAAa 71.20 1 4.01 60.16 1_7.30 11.04 1 3.29 28.59 1 8.52 NEAAb 113.17 1 9.65 117.75 1_7.65 -4.58 1 2.66 -11 86 1_5.18 BCAAc 37.36 1 1.75 32.87 1 4.68 4.49 1 2.94 11 63 + 7.62 Values are means and standard error of four steers. The (-) indicates the net release of AA. aEAA = Essential amino acids. b NEAA = Non-essential amino acids. cBCAA = Branched-chain amino acids. * Significantly different than the control group (P<.05). ' ** Significantly different than the control group (P<.Ol). Net AA uptake (or release) (m mole/hr) + 66 2 hr 4 hr Before feeding after feeding after feeding 20‘ 40. 601 $fiupvmozamfizfi $Too'cngrr; $155561; mgcs > mgca>> m>c= ; :> z: 3: Figure 6. The net amino acid uptake (or release) by the hind limb of steers fed the low protein diet. 67 mainly after feeding. When the high protein diet was fed, a complete change in the behavior of all AAs was observed. Even before feeding (Table 18 and Figure 7) there was a significant net uptake of thr, ser, ile, tyr, arg, EAA and BCAA. This observationnfight be due,in part,to a higher net availability of amino acids to the steers during the experimental period (two weeks). At 12 as well as T4, the arterial blood had higher AAs concentrations as showiin Tables 19 and 20. Almost all AAs (except asp, asn, gln) con- centrations were significantly increased in the arterial blood at either T2 or T4. Total BCAA concentrations significantly increased (85%) from 40 u mole/d1 to reach a maximum level of 76 u mole/d1 at two hours after feeding. Tables 19 and 20 also show the significantly high net uptake of all AAs (except ala, gln, asn which were released) at all times after feed- ing. The AV differences were also larger. The high net amino acid uptake generally noted in the high protein fed steers was due to .. high arterial PAA concentrations(Tables 19 and 20), as well as the high PF (Table 11) through the hind limbs. The release of ala increased in a linear pattern with time after feeding to reach a maximum of 88 m mole/hour which is more than double the amount released before feeding. Glutamine was also released. How- ever, it was released in lesser quantities than in either control- or 1 low protein-fed steers. Figure 7 shows that the high EAA uptake continued to be at the same high level at 14 (last time interval measured) as at T2, while the net uptake of NEAA at T4 decreased to almost one half of 'that noted at T2. 68, TABLE 18. The Arteriovenous Concentration Difference and the Net Uptake of Amino Acids by the Hind Limb of Steers Fed the High Protein Diet (Immediately Before Feeding) Plasma AA level (n moel/dl) Net uptake AA Arterial Venous Difference m mole/hr Asp 1.36 1_ .07 1.39 1_ .67 - .03 1_ .04 - .20 1_ .26* Thr 6.44 1_ .24 4.20 1_ .16 2.24 1_ .14 14.60 1_ .91** Ser 8.33 1. .51 4.30 1_ .24 4.03 1_ .27 26.28 1_1.76** Asn 1.40 1_ .08 1.61 1_ .65 - .21 1_ .03 -1.37 1_ .20 Glu 11.81 1_ .50 11.31 1_ .82 .50 1, .18 3.26 1 1.17 Gln 15.42 1_1.88 17.31 1 1.54 -1.89 1 1.34 -12.32 1 8.71 .Gly 16.05 1_ .60** 17.57 1_2.26 -1.52 1_ .34 -9.91 1 2.21 Ala 8.89 1_ .18 14.38 1 1.37 -5.49 1_ .20 -35.79 1 1.30 Pro 8.01 1_ .21 8.07 1 1.21 - .06 1_ .02 - .39 1, .13** Val 18.49 1_ .80 15.59 1 2.16 2.90 1_ .64 18.91 1 4.16 Cyst 2.21 1_ .07** 2.33 1, .66 - .12 1_ .01 - .78 1_ .07** Met .53 1_ .23 .64 1_ .17 - .11 1_ .06 - .72 1_ .40 Ile 8.30 1_ .31 6.92 1 1.12 1.38 1_ .19 9.00 1 1.24** Leu 13.67 1_ .36 10.86 1 1.12 2.81 1. .24 18.32 1 1.56 Tyr 3.04 1_ .11 2.56 1_ .64 .48 1_ .07 3.13 1_ .46** Phe 4.70 1_ .16 4.64 1_ .67 .06 1_ .09 .39 1_ .60 Orn 10.43 1_ .78 10.28 1 1.71 .15 1_ .93 .98 1 6.24 Lys 6.95 1_ .52 6.85 1_1.14 .10 1_ .62 .65 1_4.03 His. 5.34 1. .14 5.38 1 1.14 - .04 1_ .02 - .26 1_ .13** Arg 6.24 1_ .51 .73 1 1.06 1.51 1_ .45 9.85 1 2.93* EAAa 70.66 1 3.27 62.81 1 2.68 8.25 1 1.19 53.79 1_7.76** NEAA” 86.95 1 4.99 91.111 8.87 -4.16 11.12 -27.12 1 7.30 BCAAc 40.46 1 1.47 33.37 1_3.40 7.09 1 1.07 46.23 1 6.98** Values are means and standard The (-) indicates the net release of AA. aEAA = Essential amino acids. b error of four steers. NEAA = Non-essential amino acids. cBCAA = Branched-chain amino acids. * Significantly different than the control group (P<.05). **Significantly different than the control group (P<.Ol). 69 80‘ 2hr 4hr 60 ‘ Before feeding after feeding after feeding 40 1 20 i 0. =- _l 20 . 1: 40 - L. .C 1 E LJ 2 60 ‘ E v 80. _1 g 100 ”J J- £5 I I I I I I I I I I S. :3 w + fi II III II II 4.) S- 250 [-1 < < +. 270 33 lg! II 400]: “.1 II II II up 460 . [-1 4801 500 ' bl 59556.4: .5. age 51% e eee ‘me' ”5‘3 E; mic: g; mic: E; Figure 7. The net amino acid uptake (or release) by the hind limb of steers fed the high protein diet. 7O TABLE 19. The Arteriovenous Concentration Difference and the Net Uptake of Amino Acids by the Hind Limb of Steers Fed the High Protein Diet (Two Hours After Feeding). Plasma AA level (n mole/d1) Net uptake AA Arterial Venous Difference m mole/hr Asp 2.76 1_ .20 1.35 1_ .19 1.41 1_ .64 9.19 1. .26* Thr 11.42 1_ .27** 4.80 1. .44 6.62 1_1.19 43.16 1_1.24** Ser 15.54 1_ .20 5.65 1 1.31 9.89 1 1.71 64.48 1 7.22* Asn 1.65 1_ .02 1.79 1_ .76 - .14 1_ .14 - .91 1. .91 Glu 25.88 1_0.41** 11.64 1 1.41 14.24 1 1.98 92.84 1 6.37** Gln 14.47 1. .66 17.59 1 2.58 -3.12 1 1.18 -20.34 1 1.17 Gly 35.75 1_ .93** 14.65 1 1.13 21.10 1 2.23 137.57 1 7.99** Ala. 18.30 1_ .30** 25.50 1 3.45 -7.20 1 1.15 -46.94 1 7.48 Pro 16.08 1_ .38** 7.41 1_1.81 8.67 1_ .43 56.52 1 2.80** Val 34.32 1_ .86** 16.68 1 1.35 17.64 1_ .51 115.01 1_3.32** Cyst 4.98 1_ .20** 2.09 1_ .63 2.89 1_ .17 18.84 1.1.11** Met 1.80 1_ .16** .71 1_ .28 1.10 1_ .12 7.17 1. .78** Ile 15.78 1_ .52** 6.88 1 1.15 8.90 1_ .37 58.03 1 2.41** Leu. 26.44 1_ .62** 11.46 1_ .38 14.98 1 2.24 97.66 1 1.56** Try 6.12 1_ .10* 2.62 1_ .24 3.50 1 1.14 22.82 1_ .91** Phe 10.08 1_ .34** 4.82 1_ .49 5.26 1 1.15 34.30 1_ .98** Orn 19.20 1_ .57 8.88 1 2.69 10.32 1 1.14 67.29 1 7.41* Lys 12.80 1_ .38 5.92 1_ .46 6.88 1_1.28 44.86 1_1.82** His. 10.72 1_ .25 4.94 1_ .54 5.78 1 1.25 37.68 1 l.63** Arg. 14.12 1_ .23** 5.44 1_ .87 8.56 1 1.62 55.81 1 4.03** EAAa 134.48 1 8.63* 61.65 1 8.96 72.83 1 4.67 474.85 130.45** NEAAb 160.73 1 3.97* 99.17 1 8.60 61.56 1_4.03 401.37 126.27** BCAAC 76.54 1 2.00** 35.02 1_3.88 41.52 1_1.12 270.71 1 7.30** Values are means and standard The (-) indicates the net release of AA. aEAA = Essential amino acids. b error of four steers. NEAA = Non-essential amino acids. cBCAA = Branched-chain amino acids- * , .Significantly different than the control group (P<.05). IISignificantly different than the control group (P<.Ol). 71 4 TABLE 20. The Arteriovenous Concentration Difference and the Net Uptake of Amino Acids by the Hind Limb of Steers Fed the High Protein Diet (Four Hours After Feeding). Plasma AA level (u mole/d1) Net uptake AA Arterial Venous Difference m mole/hr Asp 2.78 1_ .08 .94 1_ .18 1.84 1_ .11 11.99 1. .72** Thr. 11.80 1_ .l4** 4.28 1_ .27 7.52 1 1.14 49.03 1_ .91** Ser 15.14 1_ .06** 4.82 1_ .53 10.32 1_1.76 67.29 1 4.94* Asn 1.58 1_ .14 1.69 1_ .65 - .11 1. .10 - .72 1. .65 Glu 23.16 1 1.36 11.06 1 1.36 12.10 1 2.17 78.89 113.65* Gln 16.82 1_ .59 19.26 1 2.26 -2.44 1_ .33 -15.91 1 2.15 Gly 36.64 1 3.43 15.35 1 1.46 21.29 1 2.02 138.81 113.13* Ala 13.66 1_ .15** 27.26 1 4.58 -13.51 1 4.43 -88.09 1 8.80* Pro 16.32 1_1.20 6.36 1_ .36 9.96 1 1.84 64.94 1 5.46** Val 29.58 1. .92** 15.32 1_2.35 14.26 1. .57 92.98 1 3.71** Cyst 4.32 1_ .08** 1.88 1_ .04 2.44 1_ .64 15.91 1_ .26** Met. 1.66 1_ .14* .39 1. .06 1.27 1. .68 8.28 1_ .52** Ile 16.20 1_1.22** 6.60 1_1.21 9.60 :_1.01 62.59 1_6.56** Leu 26.20 1 1.27** 10.73 1 2.10 15.47 1_1.17 100.86 1_7.61** Tyr 6.06 1 1.09** 2.08 1_ .67 3.98 1_ .02 25.95 1_ .13** Phe. 9.54 1_1.13* 4.10 1_ .62 .44 1 1.01 35.47 1_6.57* 0rn 20.40 1 1.50* 8.19 1_ .36 12.21 1_1.20 79.61 1_7.80* Lys 13.60 1_ .33** 5.46 1_ .26 8.14 1. .13 53.07 1_ .85** H3 10%: Ah 424:.u 5M11Jl 43m: 73* Arg 13.96 1 1.20* 4.47 1 1.16 9.49 1 1.04 61.87 1 6.76** EAAa 133.42 1 2.48** 55.59 1 1.71 77.83 1 2.77 507.45 1 5.02** NEAAb 126.77 1 2.68 98.89 1 7.74 27.88 1 4.46 181.78 129.08 BCAAC 71.98 1 1.41** 32.65 1 1.66 39.33 1 2.75 256.43 1_4.89** Values are means and standard The (-) indicates the net release of AA. aEAA = Essential amino acids. ”NEAA ”BCAA ** Significantly different than the control group (P<.Ol). error of four steers. Non-essential amino acids. Branched-chain amino acids. * . Significantly different than the control group (P<.05). 72 II. Effect of Insulin and Starvation Table 21 summarizes the data of BF, PCV and PF of another group of steers fed the same control diet (Table 4). insulin-treated, starved for 24 hours or starved for 48 hours. As observed in the first study of this series, there were no significant differences in PCV between all treatments with an overall mean of 29%. Insulin had no effect on either BF or PF, while starvation (for either 24 or 48 hours) led to a marked decrease in both. Tables 22, 23 and 24 present the AV difference and the net uptake of AAs by the hind limb of steers fed the control diet (at T0, T2 and T4 respectively). Before feeding the primary AA released were ala and to some extent, gln (Figure 8). However, at 12 as well as T4 net release was significantly (P<.Ol) decreased, especially for ala. Tables 23 and 24 show that there were no significant increases in the arterial PAA concentrations (except for gly at T2) over the time interval sampled. However, the same tables also show a significant net uptake of asp, ser, asn, gly, val, and leu, with highest net uptakes observed at T2 (Figure 8). The AA concentrations and net uptake of this control group behaved similarly to the first control group. However, AV differences and the net uptake (Figure 8) were lower than those of the first controls (Figure 5). These differences may be due to: l) in the first study there were only two steers whose cannulas stayed patent (see Materials and Methods for details), which were used as control for the first study. These steers had been fed the high-protein diet previously and hence. there may have been a carryover effect from that treatment (high- protein diet) on the control. 2) The small number of animals used 73 TABLE 21. Effect of Insulin Injection and Starvation on the Plasma Flow Rate Across the Hind Limb. Criteria Blood flow Packed Cell Vol. Plasma flow Treatment liter/hr % liter/hr Control 580 1 41.9” 28.45 1 0.95” 414 1 30.0” Insulin Injection 605 1 37.7” 29.05 1 1.32” 429 1 26.8” 24 hr Starvation 335 1 23.9” 28.90 1 0.50” 238 1 17.0” 48 hr Starvation 315 1 29.9” 29.60 1 0.67” 221 1 21.1” aValues are means and standard error of four steers. b,c different (P<.Ol). Values not sharing common superscript in column were significantly 74 TABLE 22. The Arteriovenous Concentration Difference and the Net Uptake of Amino Acids by the Hind Limb of Steers Fed the Control Diet (Immediately Before Feeding). Plasma AA level (u mole/dllf Net uptake AA Arterial Venous Difference m mole/hr Asp .98 1, .10 1.46 :_ .73 -0.48 1. .03 -1.99 1. .13 Thr 2.72 :_ .20 2.67 :_ .37 .05 :_ .07 .21 :_ .29 Ser 6.07 1_1.32 6.04 1_1.87 .03 :_ .04 .12 :_ .17 Asn .89 i_ .10 1.09 :_ .65 - .18 :_ .05 - .75 1. .21 Glu 6.26 1_ .31 5.86 :_ .46 .40 :_ .15 1.66 :_ .62 Gln 8.58 1. .70 12.31 1_1.78 -3.73 :_ .68 -15.44 :_ .33 Gly 10.46 1_ .50 12.88 1_1.75 -2.42 :_ .25 -10.02 :_1.04 Ala 8.64 :_ .08 19.97 :_2.60 -11.33 1 1.52 -45.51 1_2.15 Pro. 4.97 :_ .30 3.92 1_ .29 1.05 i. .06 4.35 :_ .25 Val. 9.45 :_ .78 8.45 1_1.15 1.00 :_ .37 4.14 1_1.53 Cyst .28 :_ 14 .61 :_ .04 .17 :_ .10 .70 :_ .41 Net. .61 1_ .14 1.21 1_1.07 - .60 :_ .93 -2.48 i 3.85 Ile 4.92 :_ .31 3.92 1_1.25 1.00 :_ .06 4.14 :_ .25 Leu 7.26 :_ .37 5.65 :_1.83 1.61 1_ .46 6.67 1_1.90 Tyr 1.95 :_ 20 1.50 :_ .68 .45 :_ .12 . 1.86 :_ .50 Phe 3.68 :_ .38 3.42 :_ .43 .26 1. .05 1.08 :_ .21 Orn 6.39 :_ .32 5.49 :_1.67 .90 1_ .29 3.72 1 1.20 Lys 4.26 :_ .21 3.66 :_ .41 .60 :_ .26 2.48 1. .82 His 3.31 :_ .20 2.61 :_ .76 .70 j_ .04 2.90 :_ .17 Arg 3.57 1_ .37 4.27 :_ .74 - .70 :1 .23 -2.90 :- .95 EAAa 39.78 i 2.96 35.86 1_4.71 3.92 1_1.75 16.23 1_7.25 NEAAb 55.97 1_4.07 71.11 1_6.11 -15.14 1_1.04 -62.70 1_4.32 BCAAC 21.63 1_1.46 18.02 1 2.23 3.61 :_1.77 14.95 i_3.19 Values are means and standard error of four steers. The (-) indicates the net release of AA. aEAA = Essential amino acids. b cBCAA = Branched-chain amino acids. NEAA = Non-essential amino acids. 75 TABLE 23. The Arteriovenous Concentration Difference and the Net Uptake of Amino Acids by the Hind Limb of Steers Fed the Control Diet (Two Hours After Feeding). Plasma AA level Lu mole/d1) Net uptake AA Arterial Venous Difference m mole/hr Asp 1.34 :_ 66 1.12 1. .02 .22 :_ .04 .91 :_ .17** Thr 3.09 1_ 38 2.56 1_ 16 .53 1_ .22 .19 :_ .91 Ser 6.79 1_1.27 -5104 1 2.14 1.75 1_ .13 7.25 1_ .54** Asn. 1.27 :_ .09 1.09 :_ 64 .18 1_ .05 .75 :_ .21* Glu 6.93 1. .19 5.90 i 1.51 1.03 1_ .32 4.26 :_1.32 Gln. 11.44 i 1.03 14.02 :_2.08 -2.63 :_ .95 -10.89 i_3.93 Gly 13.27 :_ 39* 11.52 :_2.29 1.75 :1 .10 7.25 1_ .41** Ala. 8.92 :_ .23 10.02 :_1.39 -l.05 1_ .16 -4.35 1_ .66** Pro 5.54 1_1.78 3.95 :_ 17 1.59 1_ .61 6.59 1_2.52 Val 11.09 1_1.16 8.32 1_1.20 2.77 1_ .64 11.47 1_ .17* Cyst .90 1_ .15 .65 1_ .04 .25 1_ .11 1.04 1_ .46 Met .71 1, .11 .16 :_ .06 .55 :_ .05 2.28 :_ .21 Ile 5.39 1. .61 3.74 1_ .28 1.65 1_ .83 6.83 1_1.37 Leu 7.82 :_ .37 3.30 1_ .67 4.52 :_ .80 18.71 1_1.24** Tyr. 2.05 1_ .31 1.43 1_ .11 .62 1_ .21 2.57 1. .87 Phe 3.66 :_ .38 3.27 :_ .89 .39 :_ .01 1.61 :_ .04 Orn 6.86 1_1.39 5.30 :_ .68 1.56 :_ .29 6.47 1_1.20 Lys 4.57 1_1.26 3.53 1_ .45 1.04 :_ .19 4.31 1_ .79 His 3.69 :_ .52 2.63 :_ .71 1.06 1_ .41 4.39 1_1.70 Arg 4.83 1. .36 4.28 i_ .68 .55 :_ .28 2.28 1_1.16 EAAa 44.85 1_4.15 31.79 1_3.40 13.06 1_1.75 54.07 1_3.11* NEAA” 65.36 1 4.89 60.09 1 8.47 5.27 1_1.42 21.82 :_5.88** BCAAc 24.30 1 2.14 15.36 1_2.75 8.94 :_1.70 37.01 1_7.04 Values are means and standard error of fiOursteers. The (-) indicates the net release of AA. aEAA = Essential amino acids. bNEAA = Non-essential amino acids. cBCAA = Branched-chain amino acids. *Significantly different than before feeding (P<.05). **Significant1y different than before feeding (P<.Ol). 76 TABLE 24. The Arteriovenous Concentration Difference and the Net Uptake of Amino Acids by the Hind Limb of Steers Fed the Control Diet (Four Hours After Feeding) Plasma AA level In mole/d1) Net uptake AA Arterial Venous Difference m mole/hr Asp 1.56 1_ .63 1.29 :_ .08 .27 1_ .05 1.12 1_ .21** Thr. 3.32 1_ .46 2.67 :_ .21 .65 1. .20 2.69 1_ .82 Ser 6.64 1_1.26 5.63 1_1.18 1.01 :_ .68 4.18 1_ .33** Asn 1.32 :_ .16 1.19 1_ .67 .13 1_ .09 .54 1_ .37 Glu 7.72 1_ .83 6.70 :_ .48 1.02 :_ .15 4.22 :_ .62 Gln 9.30 1_ .73 12.03 1_1.49 -2.73 :_ .24 -1l.30 1. .99 Gly 11.57 1_ .29 11.48 1 1.06 .09 :_ .23 .37 1_ 94** Ala 9.49 :_ .22 10.48 1_2.39 - .99 :_ .17 -4.10 :_ .69** Pro 4.56 i 1.33 4.10 1_ .26 .47 1_ .07 1.92 1_ .29 Val 9.49 :_ .34 7.42 1_ .59 2.07 :_ .25 8.57 :_1.03 Cyst. .67 1_ .02 .59 :_ .05 .08 1_ .08 .33 1_ 13 Met .46 1_ .03 .31 1, 06 .15 1_ .03 .62 1_ .12 Ile 4.89 1_1.29 3.88 1_ 63 1.01 1_ .26 4.18 1_1.07 Leu 7.74 1_1.21 6.16 1_ .82 1.58 1_ .61 6.54 1_2.50 Tyr. 1.98 :_ .29 1.51 :_ .69 .47 :_ .20 1.95 1_ .82 Phe 3.54 :_ .26 3.43 1_ .88 11 :_ .11 .46 1_ .45 Orn 6.44 1_ 18 5.43 1_1.65 l 01 1_ .47 4.17 1_1.93 Lys 4.29 :_ .12 3.62 1_ 48 67 :_ .31 2.78 1_1.27 His 3.04 1_ .22 2.73 1_ 17 .31 1. .05 1.28 1_ .21 Arg. 4.60 1. .29 4.50 1_ .09 .10 :_ .20 .41 1_ .82 EAAa 41.37 1_2.16 34 72 1 2.78 6.65 1_ .62 27.53 1_2.57 NEAAb 61.25 :_3.84 60.43 :_8.80 .82 1. .04 3.39 i_ .17** BCAA 22.12 :_ .84 17.46 1_1.44 4.66 1_1.60 19.29 :_2.48 Values are means and standard error of four steers. The (-) indicates the net release of AA. aEAA = Essential amino acids. ”NEAA ”BCAA Non-essential amino acids. Branched-chain amino acids. **5ignificant1y different than before feeding (P<.Ol). Net AA uptake (or release) (m mole/hr) 77 80- 2 hr 4 hr Before feeding after feeding after feeding 60 ' 40 ' 20 . 91v vvoa 0191 "19- W3 53 a. :5 5: QlV' "l9 ' VVOB. 1 53 m :1 3: elv- "l9' VVOS. VVHN VVHN“ VVHN“ Figure 8. The net amino acid uptake (or release) by the hind limb of steers fed the control diet (II). 78 (two steers in the first control vs. four in the second) along with . differences in their body weight may account_for part of the differences observed. A. Effect of Insulin When insulin was injected into the jugular vein of the steers fed the control diet, the data in Tables 25, 26, 27 and 28 were obtained. Data in Table 25 and Figure 9 show that the AAs behaved similarly at T4 for the control-fed and T0 (before injection) of insulin-injected steers. Therefore, statistical comparisons were made with T0 (before injection) and each animal served as his own control. Insulin injection had no effect on the AA concentration in the arterial blood (Tables 26, 27 and 28) but it affected net hind limb uptake of amino acids. As shown in Figure 10 there was an overall net uptake of BCAA and EAA at T1 and the uptake due to insulin at T2 of gly, phe, arg, BCAA, EAA and NEAA was significant (P<.05) (Table 27). Such an effect on AA uptake by insulin should have been observable at T1 (Table 26), but, the high standard error of those data masked ' differences. Four hour post-injection the net uptake of both EAA and NEAA started to diminish as shown in Table 28 and Figure 9; and ala release was significant. The net release of ala, gln and NEAA at T4_was similar to those at T0 of the first control group (Figure 5). B. Effect of Starvation When the steers were starved for either 24 or 48 hour a net release of almost all AAs was observed. Table 29 shows that 24 hour starvation has a slight effect on the arterial PAA concentrations. Glutamate and 1eu significantly decreased 79 TABLE 25. The Arteriovenous Concentration Difference and the Net Uptake of Amino Acids by the Hind Limb of Steers Fed the Control Diet and Treated with Insulin (Immediately Before Insulin Injections). Plasma AA level (u mole/d1) Net uptake AA Arterial Venous Difference m mole/hr Asp 1.331 .22 .941 .04 .391 .18 1.671 .77 Thr. 4.79 11.69 3.97 1 .49 .82 1 .20 3.52 1 .86 Ser 4.53 11.33 4.74 1 .90 - .211 .57 - .90 1 2.45 Asn 1.371 .62 1.45 1 .06 - .081 .04 - .341 .17 Glu 5.94 1 .90 4.95 11.70 .99 1 .20 4.25 1 .86 Gln 12.48 1 .84 14.43 1 2.12 -1.95 1 .72 -8.37 1 3.09 Gly 13.96 11.51 13.25 11.28 .711 .77 3.05 1 3.30 Ala 9.85 1 .54 11.52 11.91 -1.67 1 .37 -7.16 11.59 Pro. 6.39 11.21 6.17 1 .12 .23 1 .09 .96 1 .39 Val. 11.57 1 .43 10.95 11.63 .62 1 .20 2.66 1 .86 Cyst 1.081 .04 1.061 .67 .021 .03 .091 .13 Met 1.491 .27 .861 .27 .631 .06 2.701 .26 Ile 6.711 .25 6.48 11.62 .23 1 .37 .99 11.59 Leu 8.89 11.66 7.74 1 .45 1.15 1 .30 4.93 11.29 Tyr 3.321 .47 2.531 .86 .791 .11 3.391 .47 Phe 3.311 15 3.071 .75 .241 .05 1.031 .21 Orn 10.07 11.28 7.95 11.40 2.12 1 .72 9.08 1 .51 Lys 6.711 .85 5.301 .93 1.411 .68 6.051 .34 His .261 .74 4.111 .08 .151 .09 .641 .39 Arg. 4.971 .13 4.691 .20 .281 .07 1.201 .30 EAA” 52.7 1 3.51 47.17 1 8.82 5.53 1 0.31 23.72 11.33 NEAA” 70.32 1 5.36 68.99 1 5.96 1.33 1 .66 5.711 2.57 BCAA” 27.17 11.37 25.17 1 2.20 2.00 1 0.36 8.58 11.54 Values are means and standard error of'finnisteers. The (-1 indicates the net release of AA. aEAA = Essential amino acids. bNEAA = Non-essential amino acids. CBCAA = Branched-chain amino acids 80 TABLE 26. The Arteriovenous Concentration Difference and the Net Uptake of Amino Acids by the Hind Limb of Steers Fed the Control Diet and Treated Nith Insulin (One Hour After Insulin Injection). Plasma AA level (0 mole/d1) Net uptake AA Arterial Venous Difference m mole/hr Asp. 1.12 1_ .27 .87 1_ .12 .25 1_ .15 1.07 1_ .63 Thr 4.17 1. .96 3.87 1_1.32 .30 1_ .64 .29 1 2.69 Ser 3.69 1_ .76 2.08 1_ .85 1.61 1_ .41 6.91 1 1.72 Asn 1.38 1_ .34 1.89 1_ 74 - .51 1_ .20 -2.19 1_ .84 Glu 5.08 1 1.24 3.96 1_ .78 1.12 1_1.06 4.80 1_4.45 Gln 11.10 1 2.01 11.88 1_1.74 - .78 1_ .87 -3.35 1_3.65 Gly 11.36 1 2.11 6.99 1_1.96 4.37 1_1.15 18.75 1_4.83 Ala 9.71 1 1.97 10.16 1 1.61 - .45 1 1.01 -l.93 1 4.24 Pro 5.96 1_1.38 3.19 1_ .29 .77 1 1.69 11.84 1 4.58 Val. 9.50 1_1.73 5.67 1 1.85 83 1_1.12 16.43 1_ .50** Cyst 1.12 1_ .28 .71 1_ .06 .41 1_ .22 1.76 1_ .92 Met 1.13 1_ .29 .72 1_ .15 .41 1_ .14 1.76 1_ .59 Ile 6.17 1_1.32 4.05 1_1.59 2.12 1_ .73 9.09 1 3.07 Leu. 7.66 1_1.57 4.52 1_1.70 3.14 1_ .87 13.47 1 3.65 Tyr 2.92 1_ .65 1.49 1_ .27 1.43 1_ .38 6.13 1_1.60 Phe 2.84 1_1.55 1.76 1_ .27 1.09 1_ .28 4.68 1 1.18 Drn 8.67 1 1.86 4.65 1_1.65 4.02 1 1.20 17.25 1_5.04 Lys 5.78 1 1.24 3.10 1_ .48 2.68 1_ .81 11.50 1 3.40 His 3.97 1_ .92 2.13 1. .79 1.84 1_ .73 7.89 1 3.07 Arg. 5.01 1_1.22 3.38 1_ .42 1.63 1_ .80 6.99 + 3.36 EAAa 46.23 1 9.80 29.20 1 4.92 17.03 1 4.88 73.06 120.94 NEAAb 62.11 112.87 47.87 1_4.77 14.24 1_3.10 61.09 134.75 BCAA 23.33 1 4.62 14.24 1 3.67 9.09 1 1.48 39.00 1_6.35 Values are means and standard error ofibur steers. The (-) indicates the net release of AA. aEAA = Essential amino acids. bNEAA = Non-essential amino acids. ”BCAA Branched-chain amino acids * * Significantly different than before injection (P<.Ol). 81 TABLE 27. The Arteriovenous Concentration Difference and the Net Uptake of Amino Acids by the Hind Limb of Eight Steers Fed the Control Diet and Treated with Insulin (Two Hours After Insulin Injection) Plasma AA level (n mo1e/d11 Net uptake AA Arterial Venous Difference m mole/hr Asp 1.20 1. .68 .81 1_ .06 .39 1_ .02 1.67 1_ .08 Thr. 4.49 1_1.15 3.02 1_ .15 1.47 1_ .05 6.31 1_ .21 Ser 3.67 1_ .77 3.42 1. .14 .25 1_ .03 1.07 1_ .13 Asn. 1.44 1_ .06 1.46 1_ .67 - .02 1_ .01 - .09 1_ .04 Glu 4.62 1 1.26 3.59 1_ .27 1.03 1_ .61 4.42 1_ 04 Gln 13.21 1 1.40 13.91 1_1.63 - .70 1_ .77 -3.00 1 3.24 Gly 12.13 1_ .95 6.26 1_ .58 5.87 1_ .42 25.18 1_1.76* Ala. 9.99 1_ .44 10.08 1_1.41 - .09 1_ .03 - .39 1_ .13 Pro. 7.82 1 1.94 5.25 1_ .65 2.57 1_1.29 11.01 1_5.42 Val .84 1_ .79 4.80 1_ .87 5.04 1_ .68 21.62 1_ 34** Cyst .90 1_ .09 .52 1_ .76 .38 1_ .07 1.63 1_ 29 Met .94 1_ .16 .46 1_ .11 .48 1_ .01 2.06 1_ 04 Ile 5.14 1_ .36 2.84 1_ .65 2.30 1_ .35 9.87 1 1.47* Leu 6.42 1 1.25 3.51 1_ .38 2.91 1_ .13 12.48 1_ .55* Tyr 2.71 1_ .66 2.11 1_ .16 .60 1_ .10 2.57 1_ .42 Phe 2.62 1_ .69 1.39 1_ .14 1.23 1_ .06 5.28 1_ .25** Drn 7.91 1 1.26 6.78 1_ .51 1.13 1_ .25 4.83 1_1.05 Lys 5.27 1 1.17 4.52 1_ .34 .75 1_ .17 3.22 1_ .71 His 5.21 1 1.29 3.50 1_ .48 1.71 1_ .86 7.34 1_3.61 Arg. 5.35 1_ .21 2.78 1_ .31 2.57 1_ .16 11.03 1_ .42** EAAa 45.28 1 3.35 26.82 1 3.38 18.46 1 1.03 79.19 1_ .13** NEAAb 65.60 1 5.71 54.19 1 8.59 11.41 1 2.12 48.95 1 9.09* BCAAC 21.40 1 1.34 11.15 1_1.90 10.25 1_ 56 43.97 1 2.40** Values are means and standard The (-) indicates the net release of AA. aEAA = Essential amino acids. bNEAA = Non-essential amino acids. CBCAA = Branched-chain amino acids. error off0ursteers. * Significantly different than before injection (P<.05). ** Significantly different than before injection (P<.Ol). 82 TABLE 28. The Arteriovenous Concentration Difference and the Net Uptake of Amino Acids by the Hind Limb of Steers Fed the Control Diet and Treated with Insulin (Four Hours After Insulin Injection). Plasma AA level (u mole/d1) Net uptake AA Arterial Venous Difference m mole/hr Asp 1.93 1. .81 1.58 1_ .05 ‘ .35 1_ .26 1.50 1 1.09 Thr 3.25 1_ .12 2.64 1_ .67 .61 1_ .05 2.62 1; .21 Ser. 3.51 1 1.14 3.15 1_ .14 .36 1_ .05 1.54 1_ .21 Asn 1.42 1. .69 1.26 1. .02 .16 1_ .07 .69 1_ .29 Glu 6.83 1_ .27 6.14 1_1.02 .69 1_ .25 2.96 1 1.05 Gln 11.40 1_1.42 12.79 1 1.07 -1.39 1_ .65 -5.96 1 2.73 Gly 11.40 1_1.43 10.84 1_ .21 .56 1_ .22 2.40 1_ .93 Ala 9.36 1_ .34 19.71 1_2.25 -10.37 1_ .09 —44.49 1_ .38** Pro 4.34 1 1.18 4.02 1_ .69 .32 1_ .09 1.35 1_ .38 Val 7.71 1_ .38** 7.52 1 1.30 .19 1_ .08 .82 1. .34 Cyst .78 1_ .06 .67 1_ .08 .11 1_ .03 .47 1_ .13 Net .74 1_1.06 .66 1_ .06 .08 1_ .94 .34 1_3.95 Ile 5.06 1_ .14* 4.37 1_1.09 .69 1. .05 2.96 1_ .21 Leu 7.33 1_ .69 5.76 1 1.15 1.57 1_ .54 6.74 1 2.27 Tyr 2.22 1_ .10 1.76 1_1.03 .46 1_ .07 1.97 1_ .29 Phe 2.74 1. .12 2.40 1_ .67 .34 1_ .05 1.46 1_ .21 Orn. 5.99 1_ .18 5.30 1. .68 .69 1. .10 2.96 1_ .42** Lys 3.99 1_ .12 3.53 1. .65, .46 1_ .07 1.97 1_ .29 His. 2.89 1_ .12 2.68 1_ .96 .21 1_ .06 .90 1_ .25 Arg 5.09 1_ .14 4.66 1_1.09 .43 1_ .05 1.84 1_ .21 EAAa 38.80 1_2.83 34.22 1 2.94 4.58 1 1.89 19.65 1 8.11 NEAAb 59.18 1 2.52 67.22 1 2.99 -8.04 1_ .53 -34.49 1_2.27** BCAAC 20.10 1_1.21* 17.65 1_1.54 2.45 1 0.67 10.51 1 2.87 Values are means and standard The (-) indicates the net release of AA. aEAA = Essential amino acids. ”NEAA ”BCAA error of four steers Non-essential amino acids. Branched-chain amino acids- *Significantly different than before injection (P<.05). **Significantly different than before injection (P<-01)- Net AA uptake (or release) (m mole/hr) + 83 80 . 60 1 1 hr ' 2 hr 4 hr Before injection after injection after injection after injection 40 i 20 1 0c _— 20 1 40‘ d _1 A .601 L. 801 - __ >13: 06) NZ > wmmmz >WCD 6) m2 >w 001112 E‘E 35‘ “fig mEE'S'ig SE: '5‘ fig 32 E'S'Eg Figure 9. The net amino acid uptake (or release) by the hind limb of steers fed the control diet and injected with insulin. 84 TABLE 29. The Arteriovenous Concentration Difference and the Net Uptake of Amino Acids by the Hind Limb of 24 Hour-starved Steers. Plasma AA level (y mole/d1) Net uptake AA Arterial Venous Difference m mole/hr Asp. 1.05 1_ .73 .81 1_ .05 .24 1_ .08 .57 1_ .19 Thr. 3.53 1_ .39 3.99 1_1.25 - .46 1_ .14 -1.09 1_ .33 Ser 6.46 1.1.45 6.59 1_1.97 - .13 1_ .48 - .31 1_1.14* Asn 1.13 1_ .69 1.87 1_ .07 - .74 1_ .02 -1.76 1, .05** Glu. 4.06 1_ .45* 3.77 1_1 29 .29 1_ .16 .69 1_ .38 Mn RC61LN 16%1209 4991Ln 8601.9 Gly 11.41 1_1.60 11.33 1_1.69 .08 1_ .51 .19 1 1.21* Ala 13.25 1_ .86* 24.83 1_1.14 -11 58 1_1.28 -27.56 1. .67** Pro 5.39 1, .51 5.03 1_ .46 .36 1_ .10 .86 1_ .24 Val. 7.97 1_ .49 .49 1_ .82 - .52 1_ .38 -1.24 1_ .78** an. 981.m .%1 64 -J01.m -241.m Met .61 1_ .21 .54 1_ .16 .07 1_ .05 .17 1_ .12 Ile 3.69 1, .27 3.77 1_ .37 - .08 1_ .16 - .19 1_ .24* Leu 5.09 1_ .31* 6.06 1 1.22 - .97 1_ .09 -2.31 1_ .21** Tyr. 1.71 1_ .08 1.86 1_ .67 - .15 1. .01 - .36-1_ .03* Pm 3B1 03 3601L% -.n1 J2 -.%1 29 0rn 6.36 1, .17 5.34 1 1.56 1.02 1_ .39 2.43 1, .93 Lys 4.24 1, .11 3.56 1_ .87 .68 1_ .26 1.62 1, .62 His 3.59 1_ .34 3.35 1 1.27 .24 1_ .07 .57 1_ .17 Arg 4.92 1_1.79 4.47 1_1.29 .45 1_1.50 1.07 1_3.56 EAAa 36.77 1 4.04 37.93 1_3.00 ~1.16 1_1.64 -2.76 1 2.48** NEAA” 63.86 1 7.68 78.36 1_6.78 -14.50 1 0.96 -34.51 1_2.14** BCAA” 16.75 1_1.07 18.32 1_1.41 -1.57 1_ .04 -3.74 1_ .88** Values are means and standard error of finn‘steers. The (-) indicates the net release of AA. aEAA = Essential amino acids. bNEAA = Non-essential amino acids. cBCAA = Branched-chain amino acids. *Significantly different than fed steers (P<.05). *fSignificantly different than fed steers (P<.Ol)-- 85 while ala concentration increased (P<.05). When the animals continued starvation to 48 hour asp also decreased (Table 30). At 24 hour starvation there was a significant:, net release of ser, asn, gly, ala, val, ile, 1eu and tyr. In addition to those, the hind quarter started to release even more AAs after 48 hour starvation. These AAs were thr, phe, orn, lys, his, arg and met. As shown in Figure 10,after 24 hour starvation more NEAA were re- leased, while after 48 hour starvation the hind limb started to release the EAA in higher rate. The BCAA released accounted for 100% of the total EAA released at 24 hour starvation, while it accounted for only 42% after 48 hour starvation. 86 TABLE 30. The Arteriovenous Concentration Difference and the Net Uptake of Amino Acids by the Hind Limb of 48 Hour—Starved Steers. Plasma AA level (u_mole/d11 Net uptake AA Arterial Venous Difference m mole/hr Asp. .97 1_ .03* .80 1_ .63 .17 1_ .10 .38 1_ .22 Thr 3.45 1_ .36 4.42 1_1.47 - .97 1_ .11 -2.14 1_ 25* Ser 5.15 1_ .28 5.67 1_ .73 - .52 1_ .15 -1.15 1. 34** Asn 1.15 1_ .07 1.94 1_ .63 - .79 _1 .04 -l.75 1_ 09** Glu 4.15 1_ .21** 3.17 1_ 54 .98 __ .38 2.17 1_ 74 Gln 9.19 1 1.06 10.84 1_1.64 -1.65 1_ .42 -3.65 1_ 94 Gly 10.79 1_ .92 12.28 1_2.70 -1.49 .22 -3.29 1_ 48** Ala 12.76 1_ .30** 23.45 1_3.35 10.69 + 1.05 -23.62 1 1.11** Pro 4.77 1_ .51 5.19 1 1.36 - .42 + .15 - .93 1_ 34 Val 8.21 1_ .54 9.59 1_1.62 -1.38 1_ .08 -3.05 1_ .18** Cyst 91 1_ .04 1.15 1_ .65 - 25 1_ .01 - .53 1_ .03 Met. .55 1_ .03 .77 1_ .63 - .22 1_ .03 - .49 1_ 07** Ile 4.11 1_ .40 5.14 1_ .23 -1.03 1_ .77 -2.28 1_ .38* Leu 5.53 + .45* 6.35 1_ 79 - .82 1_ .26 —1.81 1_ .59** Tyr 1.72 + .08 2.13 1_ 69 - .41 1_ .01 - .91 1' .03 Phe. 3.19 1_ .24 3.90 1_ .68 - .71 1_ .16 -l.57 1. .36** Orn 6.36 1_ .56 8.10 1 1.48 -1.74 1_ .08 -3.84 1_ .l8* Lys. 4.24 1_ .37 5.40 1_1.32 -1.16 1_ .05 -2.56 1_ 11** His. 3.18 1_ .34 3.46 1_ .24 - .28 1_ .16 - .62 1_ .22* Arg. 4.82 1_ .24 5.95 1_ .26 -l.13 1_ .62 -2.50 1_ .05* EAAa 37.28 1 2.97 44.98 1 2.44 —7.70 1 1.53 -17.02 1 1.17** NEAAb 57.92 1 4.06 — 74.72 1.3.50 16.80 1_2.56 -37.l3 1_1.24** BCAAc 17.85 1 1.39 21.08 1_1.64 -3.23 1_ .35 -7.14 1_ .77** Values are means and standard error of fixnisteers. The (-) indicates the net release of AA. aEAA = Essential amino acids. b NEAA = Non-essential amino acids. cBCAA = Branched-chain amino acids. *Significantly different than fed steers (P<.05). ** . Significantly different than fed steers (P<.Ol). f f,.£\ G F. l,ifFI flih\ fCIIIHc-rlt1lsl Net AA uptake (or release) Unmole/hr) + 80 60 40 20 20 40 60 80 87 24 hr 48 hr Fed starved starved - 1_J > WEI->55 ”12 > @536) m2 >W 0551712 32:35; 67923;; EEE§§E > Figure 10. The net amino acid uptake (or release) by the hind limb of starved steers. DISCUSSION AMINO ACID METABOLISM STUDIES EXPERIMENT TWO I. Plasma Flow During the blood flow measurement, all animals (except the starved ones) were on feed. The overall average amount eaten was 64% (of the total feed) within the three hours of blood flow measurement. The literature contains very little information on the plasma flow across the hind limb of steers under different physiological states. Data in Tables 11 and 21 shows that blood flow (BF) and plasma flow (PF) were affected by the different treatments used in this study. Blood flow significantly decreased on low protein diet and starvation, while insulin injection had no effect. The high protein diet increased the BF and PF. However, due to the high standard error, a statistical difference could not be ascertained. Bell ggngl, (1975) reported that cold stress increased hind leg plasma flow in steers three- to five-fold. However, their PF values were two to three times lower than PF obtained in this study. Differences way be due to the markers (Para amino hippuric acid vs indocyanine green dye), methods used, breed, age, body weight or diets. Unlike the data in Table 21, Heitmann and Bergman (1978) found no significant difference in PF across the hind quarter between fed and 88 89 fasted sheep. They also reported that the blood flow hada high measurement error (about 4%). When the AV differences are coupled with the high rates of blood flow (about 4% error), the overall error in the calculation could be 30-40% (Heitmann and Bergman, 1978). The PF rate across the hind quarter observed by Heitmann and Bergman (1978) was equal to the portal PF rate obtained by Prior g1_§1, (1981). Since Prior _1_§1, (1981) used the same marker and methods as in this study, they obtained portal PF values of fed steers equal to those in Table 11. They also reported that the PF of concentrate—fed steers and sheep was higher than of hay-fed animals. The PCV value obtained in the present study averaged 29% and is in agreement with that obtained by Bell et 1. (1975) who reported range of 28 to 31%. II. Effect of Dietary Protein The maintenance of body protein economy is the integrated result of various interactive process affecting each of the individual AAs. Be- cause the metabolism of each AA is, in turn, regulated bya specific con- trol mechanism, understanding whole-body protein dynamics requires knowledge of the individual AA kinetic changes induced by physiological and pathological events. Examination of AA-exchange across the deep tissues of the human forearm demonstrated that in normal man in the postabsorptive state (i.e. following a 12 to 14 hour overnight fast) there is a net release of AAs from muscle tissue, as reflected by consistantly negative AV differences (London g1.gl,, 1965; Pozefsky g; 31,, 1969; Felig §1_31,. 1970). This also exists in steers as shown in Tables 12, 15, 18 and 22. E""__—_§ 90 The pattern of this release is quite distinctive wiU1 output of ala and gln exceeding that of all other AAs and accounting for over 50% of the total AAs released (Figures 5,6). This agreed with the work of Felig g1 .11. (1970) in man and Ballard §£_gl, (1976) in sheep. As shown in Tables 13, 14, and Figure 5 the arterial plasma and net uptake of most individual AAs were significantly (P<.05) increased after feeding. This was mainly due to the energy availability and the increased production of volatile fatty acid in rumen. Propionate and butyrate sti- mulate the release of insulin (Forbes,1980), which in turn stimualte the net AAs uptake and incorporation into muscle protein. This would cause a decrease in the venous PAA concentrations. Bergen (1978) reported that the jugular PAA concentrations after feeding remain unchanged or actually tend to decline. Bell g1_gl, (1975) reported that after feeding most individual AA concentrations increased in the arterial plasma, these being significant for arg, thr, gln, tyr, phe, ala and met. Bell Einél' (1975) further reported that net exchanges of individual AAs across the leg were variable, and, with the exception of arg and glu, feeding caused no significant changes. The second control group (Figure 8), however, showed lower AV dif- ferences and net uptake than those obtained for the first group (Figure 5). The differences may be due in part to the small number of animals used along with the differences in body weight. Also there may have been a carryover effect from the previous adjustment diet (high protein) for control one, but the steers were adjusted to the control diet for 14 days. The various responses in AA metabolism to alterations in dietary 91 protein intake of the fed steers are graphically displayed in Figures 5, 6 and 7. For this purpose, the 12% CP (Figure 5) diet was used to represent,more or less, recommended protein reqUirements (NRC 1976) as the reference level for comparison of the results. At this level there was a net uptake of both NEAA and EAA. The net uptake of BCAA accounted for 30% of the EAA at 12 to 50% at T4. The AA release was due to ala and gln. These observations are similar to the results obtained by Motil ggugl. (1981) with man. Arteriovenous AA concentration difference and net uptake by the hind limb of steers fed the low protein diet be- haved almost the same way as the starved animals (Figure 10). The only net uptake was of EAA. The BCAA accounted for 35 to 42% of the total EAA uptake. The net release was of ala and gln which accounted for 100% of NEAA released in this case. Working with young men Motil g; _1. (1981) concluded that at sub- maintenance level of dietary protein the reduction in 1eu flux during the postabsorptive phase was due to a reduction in both the rate of 1eu incorporation into protein as well as 1eu oxidation. The protein ingestion resulted in increased level of AAs in the circulation of nonruminant (Felig, 1975). However, in ruminant the plasma amino acid (jugular samples) concentrations after feeding tend to remain unchanged or slightly decline (Bergen, 1978). Tables 19, 20 showed that the AA concentration in the arterial plasma increased about two folds reached its peak at two hours after feeding. There were no changes in the venous PAA concentrations. When the high CP diet was fed the EAA (Mainly BCAA) remained elevated for four hours, while the NEAA started to decline within the four fours after feeding. With respect to the effect of protein intake on muscle AA exchange, 92 studies in the rat have demonstrated a net uptake by peripheral tissues of the BCAA in the absorptive period (Yamamoto §1_gl,, 1974). Studies in normal human subjects show the same trend (Felig, 1975). Figure 7 shows the net AA uptake of hind limb of high protein-fed steers observed in the present study. Compared to control-fed steers, the net uptake of EAA increased eight fold at two hours after feeding with a 15 fold increase in BCAA. At two hours after feeding NEAA increased six fold. At four hours after feeding the net uptake decreased to five fold for EAA as well as for BCAA, while it decreased to 1.6 fold for NEAA. In contrast, ala output from muscle continued to reach a peak of 88 m mole/hr at four hours after feeding. Bergman and Heitmann (1978) re- ported that ala was continuously released from the peripheral tissues in fed as well as fasted sheep to meet the liver requirement of the main gluconeogenesis precursor, ala. Aoki _1 31, (1973) and Yamamoto §1_gl, (1974) reported that-ala release continued unchanged or is reduced for only one hour after protein diet has been given to young man. From the conclusion of Felig (1975) and the data observed here, it is clear that ala playsa major role in moving the alpha amino nitrogen from muscle in the fed as well as the fasted state. III. Effect of Insulin Insulin is essential for the normal metabolism of carbohydrate, lipids and protein in the monogastric animals (Tepperman, 1968), however, 'ts role in the normal metabolism of ruminants is poorly understood. Since alloxan-induced diabetes produced a similar response in ruminant and nonruminant animals (Reid g1_gl,, 1963) insulin is apparently ' important in Carbohydrate metabolism by ruminants. Additional evidence 93 supporting an important role of insulin in ruminant animals was obtained when insulin release from the pancreas was shown to occur following an elevation of the blood levels of glucose and fructose (Manns and Boda, 1967) and of propionic and butyric acids (Manns g1_gl,, 1967 and Forbes, 1980). There were also some evidence of the role of insulin in AAs metabolism. The present data (Tables 25 to 28) show that arterial PAA was slightly (but not significantly) lower after insulin injection. The total AA re- duced to 86% of the initial level. Similar results were obtained with sheep by Call _1_gl, (1972). Their average reductions were 83% for NEAA and 66% for the EAA. Insulin is believed to increase protein synthesis in muscle (Bergen, 1974; Trenkle, 1974). Following a stimulation of protein synthesis by substrate availability (energy and AAs) and/or endocrine influences, the increase in net AA uptake is most likely. Figure 9 shows that insulin does have a significant effect on the net AA uptake by the hind limb in steers. The net uptake increased three fold for EAA, eight fold for NEAA and five fold for BCAA. These increases remained for two hours. At four hours post-injection the net uptake of EAA decrease, while the NEAA released in net amounts. Ala and gln accounted for all the NEAA released. Insulin has the same effect on the net AA uptake in the non-ruminant animals (Felig g1_gl,, 1975; Hutson §1_§1,, 1980). Bergman and Heitmann (1978) concluded that insulin had no effect on net hepatic removal and concentrations of AAs. Insulin did, however, decrease the concentrations of the BCAA indicating increased protein synthesis in muscle. Addition of insulin to the perfused rat hind limb (Grubb, 1976) resulted in a significant increase in the rate of glucose uptake and de 94 novo synthesis of ala in ruminants vs nonruminants. The proposed mechanism of insulin in stimulating the net AA uptake may be due to stimulation of AA transport (Felig, 1975; Etherton, 1982), due to a slowing of the rate of AA oXidative decarboxylation (Hutson e1_‘ 11,, 1980), or protein degradation (Young, 1980). IV. Effect of Starvation A comparison between the levels of arterial PAA in fed and starved steers showed only slight changes for most AA (Tables 29, 30). However, glu, ala and 1eu were significantly reduced. The arterial concentration and the changes upon starvation are in accord with other data on blood AAs in sheep (Wolff g1_gl,, 1972; Bergman g1_§1,, 1974; Ballard g1_§1,, 1976). However, Bergman and Pell (1982), indicated that 1eu concentra- tions increased in the arterial plasma of starved sheep. They concluded that this rise in blood 1eu concentration in starved sheep occurred because net 1eu production by peripheral tissues overcompensated for the negligible 1eu absorption by the portaledrained viscera. In comparison to sheep blood (Ballard g1_gl,, 1976), steer blood has much lower concentrations of all AAs. Bergen (1979) reported the same results in comparison between sheep and steers. Ser and ala exist in higher concentration in our studies. Phe level appears to be in similar amount in both species. There were marked differences in the net release of AAs by the hind limb when starved steers were compared to fed steers (Figure 10). In fed steers, there was an approximate balance across the hind limb with respect to total AA, while asp, ser, asn, gly, val, and 1eu are taken up by the hind limb in significant amounts and there was a net release of ala and gln. With starvation (for 24 and 48 hours), however, there was a large 95 overall negative AV difference and net release (Figure 10) for all AAs across the hind limb as expected. Comparison with data on other species is complicated by the general use of plasma rather than blood for AA measurement (Ballard ggugl., 1976) as well as the difficulty of defining equivalent conditions in monogastric to the fed and starved steers. Nevertheless, the ala AV difference of about 106 n mole/m1 in starved steers is much higher than the value re- ported for sheep (26 n mole/ml) (Ballard £1_g1,, 1976) and is equal to the value of approximately 100 that have been reported for the human fore- arm or leg (Pozefsky g1“21., 1969; Felig g1“§1., 1970). However if those values are corrected to the metabolic body weight (kg .75) it would be 1.4 n mole/kg .75 for steers, 1.38 n mole/kg .75 for sheep and 4.1 n mole/ kg .75. With this correction one can see that human blood has higher ala AV difference than steers or sheep. Does this negate the generality of the proposed ala cycle or is it perhaps an adaptation in steers (the present study) or sheep (Ballard ggugl., 1976) to account for the restricted supply of glucose? This question needs further work to be answered. It seems that the ala cycle concept must be somehow modified to encompass the ruminant data. It has been proposed that the amino group of ala is derived not only from ala per se, but also from the BCAA (see the litera- ture review for details). The residual o-keto acids corresponding to these AAs were presumed to be degraded in muscle (Krebs, 1972; Odessy g1_ 11,, 1974). However, Hutson and Harper (1981) clearly showed that the BCKA were released from skeletal muscle and accumulate in the perfusion medium and not catabolized in the muscle. The comparison between the present data plus Ballard ggngl. (1976) to the results of Pozefsky §1_al, (1969) and Felig g§_gl, (1970); who 96 worked with man, suggests that either the restricted availability of glucose carbon reduces the amount of pyruvate available to be transa minated to ala, or, the oxidation of lipid substrates spares AA oxidation (Ballard g1_g1,, 1976). Further studies are required to clarify these possibilities. CONCLUSION Based on the data obtained in these studies, the following conclusions can be made: 1. Plasma amino acid response curves are a short term procedure to study AA requirements in growing cattle when long term studies are impossible or too expensive. The total sulfur amino acid requirement was 17.82 g/d; at least 42% of the TSAA needs must be supplied by methionine. Cysteine can supply part of the TSAA needs and can spare methionine in growing steers. The esophageal groove reflex is useful for rumen bypass of liquid diets containing protein and supplemental amino acids and could be applied to sutdy AA requirements of young rum- inants with developed rumens. A low protein diet as well as starvation reduced the plasma flow across the hind limb in steers. High protein diet as well as insulin injection had no effect on plasma flow across the hind limb in steers. Arterial concentrations of almost all AA increased after feeding in growing steers. Alanine and glutamine were continously released for all treatments before as well as after feeding in growing steers. The arterial AA concentrations of the low protein — and control- steers were the same, while they were increased in the high protein-fed steers. 97 10. ll. 12. 98 The net AA uptake in the hind limb was decreased in the low protein-fed steers, while it was increased in the high protein- fed steers comparing to the control-fed steers. Insulin had no effect on the AA concentration in the arterial blood, but the hormone significantly increased the net uptake of AA in the steers hind limb. Starvation for 24 hours or 48 hours caused a net release of almost all AAs from the steers hind limb. APPENDIX A APPENDIX A DETERMINATION OF PARA-AMINO HIPPURIC ACID One vol. of whole blood was added to 12 vol. of trichloroacetic acid (10% wv) and filtered through No. 42 Whatman paper. 10 ml filtrate is heated in a boiling water bath for one half hour. A known quantity of PAH was added to appropriate volume of whole blood and used for recovery calculation. 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