Studies on the secretion of amino acids and of urea into the gastrointestinal tract of pigs. 3. Secretion of urea determined by continuous intravenous infusion of 15N-urea.

Three pigs, of 34 kg live weight, were each fitted with re-entrant cannulas both in the duodenum and terminal ileum and catheters in the jugular vein and in the carotid artery. Pigs received a diet based on wheat and dried skimmed milk in equal amounts at 12 h intervals. During the preliminary period the digesta flowing from both duodenal and ileal cannulas were collected over 12 h after feeding on two consecutive days and half of them were reintroduced into the gut and half were stored at -20 degrees C. During the experimental period 15N-urea was infused into the jugular vein for 12 hours starting with the morning meal. Total amount of urea infused was 5 g containing 1.22 g 15N-excess. The digesta from both proximal duodenal and ileal cannulas were collected and stored, while the digesta from the preliminary period were reintroduced into the respective distal cannulas. Blood samples were taken at different time of infusion. At the end of infusion period the animals were sacrificed and samples of the contents of the digestive tract and tissues were taken. Urea flux calculated according to atom-% 15N-excess of urea N in plasma was 1.23 to 2.37 g/kg body weight/day. In the duodenal digesta 94.5 +/- 0.2 and in ileal digesta 57.1 +/- 7.39 per cent of 15N were in the TCA soluble fraction. The total amount of 15N in the duodenal digesta was 1.7 to 6.3 times greater than in the ileal digesta. Only small amount of 15N was found in the caecum and almost none in the contents of colon and rectum. It is concluded that urea is secreted into all parts of the digestive tract, the main sites of urea secretion being pancreatic juice and/or bile as well as the small intestine. The total amount of urea secreted is assumed to be similar to the daily urea excretion.

Studies on the secretion of amino acids and of urea into the gastro intestinal tract of pigs. 2. Net secretion of leucine into the small and large intestines.

The experiment was carried out on 3 pigs of 34 kg body weight equipped with re-entrant cannulas both in the duodenum and terminal ileum. Furthermore, catheters were inserted into the external jugular vein and into the carotid artery. During a preliminary period digesta from both cannulas were collected and stored. This digesta was than reintroduced during a 12 hours period of intravenous infusion of 14C-leucine while outflowing digesta from the proximal parts of the cannulas were collected, stored and analyzed for leucine content, 14C-radioactivity and specific radioactivity of leucine. This paper reports the net secretion of 14C-activity and of 14C-leucine by the small intestine and the recovery of both in the content of the digestive tract at the end of the infusion. It was concluded that endogenous leucine enters the lumen of the small intestine through the intestinal wall mainly in a protein bound form. Free leucine is secreted, however, only in small amounts. Metabolites of leucine were also secreted into or formed in the lumen of the small intestine. A proportion of endogenous leucine was reabsorbed during the passage of digesta along the small intestine.

Studies on the secretion of amino acids and of urea into the gastro intestinal tract of pigs. 1. Secretion of leucine into the stomach and upper part of the duodenum during continuous intravenous infusion of 14C-leucine.

Three pigs of 34 kg live weight were fitted with a re-entrant cannula in the duodenum, and with two catheters placed in the jugular vein and carotid artery. They were fed 1.2 kg/d of wheat-dried-skimmed milk diet. Digesta from the proximal duodenal cannula were collected for 12 h on 2 consecutive days; 50% were reintroduced into the respective distal cannula and 50% were stored at -20 degrees C. Three days later 14C-leucine was infused into the jugular vein for 12 h, starting with the morning meal. During this period the digesta from the proximal cannula were collected and stored for analysis while the digesta collected previously were reintroduced into the distal cannula. Blood samples were taken from the carotid artery. The total flow of duodenal digesta in 12 h was 5760 +/- 530 g. On average 70 percent of the radioactivity in digesta was associated with the TCA-precipitable fraction. During hours 0-3 and 11-12 of infusion 60-70% and 96-98% of the radioactivity in the TCA precipitable fractions was in leucine. In the TCA soluble fraction only 30-40 per cent of the radioactivity was associated with leucine. At the plateau 2.1 and 3.3% of infused 14C-leucine and of radioactivity were recovered in the duodenal digesta. The calculated amount of endogenous protein passing the duodenum was 20.4 g/d/pig. The results are discussed in relation to previous studies on protein synthesis and secretion in which 14C- and 15N-amino acids were used.

[Action of proteinase inhibitors. 4. Effect of short-term application of chymostatin on nitrogen metabolism in the digestive tract and protein metabolism in tissue]. / Untersuchungen zur Wirkung von Proteinaseinhibitoren. 4. Mitteilung Einfluss einer kurzzeitigen Applikation von Chymostatin auf den N-Umsatz im Verdauungstrakt und den Gewebeproteinumsatz.

Chymostatin is an effective inhibitor of intracellular proteinases in vitro. In the present experiment male rats were injected intraperitonealy during a 3 days period twice daily with a solution containing 0,9 mg Chymostatin per 100 g live weight. Reference animals received a control injection containing the same solvents but no chymostatin. During this period a daily nitrogen balance was made and metabolic faecal nitrogen and true digestibility of nitrogen were estimated using 15N-labelled animals. Furthermore, apparent biological half lives of proteins in liver and intestinal tissues were determined following the decay curves for radioactivity in proteins 48 hours after injection of L-[5-3H]-arginine und L-[guanido-14C]-arginine. The fractional rate of protein synthesis in tissues was measured by a 6 hours continuous infusion technique with L-[U-14C]-tyrosine and L-[U-14C]-leucine. Among the parameters estimated only the apparent biological half lives of proteins in liver and intestinal tissues were influenced by chymostatin. However, the prolonged half lives seemed to be rather an effect of an increased reutilisation of amino acids resulting from the intracellular protein breakdown than a decreased rate of protein degradation. The in vivo effect of the proteinase inhibitor was by far inferior compared with the action in vitro. Factors like distribution, degradation and excretion of the inhibitor could be responsible for the moderate in vivo action of chymostatin.

Investigations on the amino acid secretion and absorption in the stomach of the growing pig.

Two pigs of 35 kg live weight were fitted with re-entrant duodenal cannulas anterior to the pancreatic duct and fed barley-soya or casein-wheat starch diets prior to the experiment. After 24 hours fasting they were given a single meal of 15N-labelled free amino acids-wheat starch (A) or 15N-labelled wheat (B) diets and digesta leaving the stomach was collected during 12 hours. The proportion of TCA soluble N in total N of digesta increased with time from 10 to 40% with diet B and decreased from 90 to 47% with diet A. Total N leaving the stomach within 12 hours accounted for 96% and 106% of N ingested on diet A and B, while the amount of 15N accounted for only 66 and 86% of 15N given in diets A and B, respectively. The content of endogenous N in the digesta was 1.22 g/12 h after feeding diet A and 1.67 g/12 h after feeding diet B. It was concluded that considerable amounts of N are secreted and absorbed in the part of the digestive tract proximal to the opening of the pancreatic duct.

Studies on the range of tissue protein synthesis in pigs: the effect of thyroid hormones.

The effects of thyroid hormones on the range of tissue protein synthesis in growing pigs using the constant infusion technique with [14C]lysine were studied. 2. During a 6 h infusion, samples were taken from blood and, at the end of the infusion, from liver, pancreas, stomach, small and large intestines, kidney cortex, kidney medulla, muscle and skin. 3. Lower relative specific radioactivities of free leucine and lysine in several tissues were observed in the hormone-treated group than in the untreated one. 4. The range of protein synthesis rate and the daily amount of protein synthesized in tissues was higher in all tissues after application of thyroid hormones. 5. Assuming that the organs analysed represented 70% of the total trichloroacetic acid-precipitable protein of the pig, the estimated range of daily protein synthesis was 251-490 and 312-880 g in untreated and hormone-treated pigs respectively.

Flow of endogenous and exogenous amino acids along the gut of pigs.

Digesta were collected from 5 pigs of 33 kg live weight fitted with re-entrant cannulas in the duodenum (within 20-30 cm of the pylorus) and terminal ileum. The pigs received a diet of barley, soya bean oilmeal and a vitamin and mineral mixture. The flow rates of digesta, total nitrogen and the individual amino acids were measured at different time after feeding and during two 24 h periods. A marked increase in the flow of digesta, nitrogen and amino acids was seen in the duodenum after feeding. Total flow during 24 h of nitrogen and amino acids except His, Val, Leu, Phe and Met exceeded intake. Output of nitrogen and amino acids from the duodenal cannula was 117 and 108% of intake, respectively. A method to calculate the ratio of endogenous amino acids in digesta based on the amino acid composition of digesta, diet and endogenous secretions was developed. The calculated amounts of endogenous amino acids passing the proximal duodenum and terminal ileum were 32.2 and 21.9 g per 24 h, respectively. The greatest amount of endogenous amino acids passed through the duodenal cannula in the first two hours after feeding (2-3 g/h) and then gradually decreased to 1 g per hour. The results are discussed in relation to other studies on the secretion of endogenous protein and its amino acid composition.

Effect of thyroid hormones on the in vivo protein synthesis in rats.

The studies were made in order to estimate the rate of tissue protein synthesis in euthyroid, hyperthyroid and hypothyroid rats (110 g body weight). Hyperthyroidism was simulated by daily oral application of 4,44 micrograms thyroxine (T4) and 0,89 micrograms triiodothyronine (T3)/100 g body weight. Hypothyroidism was induced by oral application of 17,8 mg of methylthiouracil (MTU) per day and 100 g body weight. In both cases the treatment lasted for 7 days. The concentration of both hormones in blood serum was twice that of the control group in the T3 + T4 treated group. The MTU application produced a decrease in T3 and T4 concentration to levels below the lower detection limits of the methods used. The fractional rate of tissue protein synthesis was estimated using the constant intravenous infusion technique with L-14C-leucine and L-14C-lysine simultaneously. The range of the fractional rate of protein synthesis was found to be higher in hyperthyroid than in control animals in all organs under study (liver, small intestine, large intestine, gastrocnemius muscle and pancreas). Protein synthesis was stimulated to the greatest extent in muscle (4,5--5,1% . d-1 in controls vs, 5,5--6,7% . d-1 in hyperthyroid animals). The influence of hypothyroidism on the rate of protein synthesis was uncertain.

[Action of proteinase inhibitors in rats. 3. Influence of leupeptin on the rate of protein synthesis and the intracellular protein degradation by use of a test system including constant infusion of labelled amino acids, estimation of 3-methyl-histidine excretion and a triple-labelling technique]. / Untersuchungen zur Wirkung von Proteinaseinhibitoren bei Ratten. 39 Mitteilung. Einfluss von Leupeptin auf die Proteinsyntheserate und den intrazellulären Protein abbau unter Anwendung eines Testsystems, das die Dauerinfusion markierter Amino säuren, die Bestimmung der 3-Methylhistidinausscheidung und eine Dreifachmarkierungstechnik einschliesst.

Male Wistar rats (initial body weight 90 g) were fed ad libitum a whole-egg diet containing 10,5% crude protein. The animals of the experimental group received in each case of 1 mg leupeptin per 100 g of body weight in 12 hrs-intervals by i. p.-injection (3 days of treatment). Control animals got a leupeptin free solution. In addition, lysine dihydrochloride-alpha-15N was applied during the first three days of experiment to all animals and the nitrogen balance was determined. Urine from the N-Balance collection was analysed for 3-methyl-histidine excretion in order to calculate the degradation rate of myofibrillar proteins. On the fourth day the fractional rate of protein synthesis in several organs was estimated using the continuous infusion technique with 14C-leucine and 14C-lysine. The apparent biological half-lives of tissue protein were determined by a triple labelling technique, with (14C)-guanidino-L-arginine, L-5-3H-arginine and 15N-Lysine. The short-term treatment 3 days) with leupeptin did not affect the weight gain, the apparent digestibility of nitrogen and the N-balance. The fractional rate of protein synthesis was highest in the small intestine followed by the large intestine, liver and skeletal muscle and no influence of leupeptin treatment was observed. Furthermore no differences in the degradation rates of myofibrillar proteins between treated and untreated animals were found. The 3-methyl-histidine excretion via urine was 1.44 mg . kg-1 day-1 in both groups corresponding to a fractional rate of degradation of myofibrillar proteins of 2,5% per day. Apparent half-lives of tissue proteins in the small intestine, large intestine and liver, respectively, were shortest when estimated from the decay curves for the 14C-label and longest from the curves for the 15N-label. Leupeptin treatment resulted in prolonged apparent half-lives of the proteins in the large intestine and of the slowly turning over proteins in the liver. However, this effect seems to be caused rather by an increased reutilization of labelled amino acids than by a decreased protein degradation. Before continuing this kind of work the rate of uptake of injected leupeptine into tissues has to be investigated. Studies dealing with the in vivo action of proteinase inhibitors on protein metabolism have to include estimations of N-balance, protein synthesis rate, intracellular degradation rate of proteins as well as amino acid reutilization.

Estimation of rate of protein synthesis by constant infusion of labelled amino acids in pigs.

1. The fractional synthetic rates of tissue proteins were studied in growing pigs using the constant-infusion technique of tracer-labelled amino acids ([14C]leucine and [14C]lysine) and the mathetmatical model for calculation, employed in rats by Garlick, Millward & James (1973). 2. During a 6 h infusion, samples were taken from blood and muscle and at the end of the infusion from liver, muscle, pancreas, heart, duodenum, jejunum, ileum, colon, and skin. The specific radioactivity of free and protein-bound leucine and lysine was estimated. 3. A quasi-steady-state in the specific radioactivity of free plasma leucine and lysine was reached within approximately 2 h, the rate-constants being 35 and 48/d respectively. 4. The specific radioactivity of free leucine and lysine in plasma was used to calculate the flux of these amino acids. It was found to be higher than the daily intake. 5. The average fractional rate of protein synthesis in muscle and heart 8.1% d, in small and large intestine the values were 50 and 33% d respectively and in liver and pancreas more than 100% d. 6. The calculation of protein synthetic rate in pig tissue using the constant-infusion method of labelled amino acids seems to be a suitable toof for study of this species.

[Mode of action of proteinase inhibitors in rats. 2. Effect of leupeptin on N-balance and biological half-life in ad lib feeding]. / Untersuchung zur Wirkung von Proteinaseinhibitoren bei Ratten. 2. Mitteilung. Einfluss von Leupeptin auf die N-Bilanz und die biologische Halbwertszeit der Darmproteine bei ad libitum-Fütterung.

During a 15-day period, growing Wistar rats (75 g) were fed ad lib. a whole-egg diet containing 10% crude protein (control group) and the same diet + a daily subcutaneous injection of 1 mg of leupeptin per animal (experimental group), respectively. To investigate the influence of the peptide aldehyde leupeptin on the M-metabolism, N-balance trials were carried out between the 1st and the 6th, and the 10th and the 15th experimental days. Leupeptine was not found to influence the true N-digestibility. However, the intermediary N-utilization, characterized by the biological value of the dietary protein, deteriorated in comparison with the control group. The peptide aldehyde under study resulted in an extension of the biological half-life of the proteins in the tissue of the small and large intestine. It was measured via the decline of radioactivity in protein following application of L-guanidino-14 C-arginine and L-(4,5-3H)-leucine. The amino acid utilization appears to have been increased as well. In connection with previous studies (SIMON et al., 1976) i, which, under somewhat different experimental conditions, leupeptine resulted in a higher intermediary N-utilization, the mentioned agent is supposed to be able to influence N-utilization. However, additional studies are required to make statements regarding the influence of dosis and metabolic situation.

Studies on the in vivo-action of leupeptin on the nitrogen retention in rats.

A daily s.c. injection of 0.5 mg leupeptin for 7 days does not influence the digestibility of food-N but seems to improve the N-retention. A daily injection of 1.0 mg leupeptin for 14 days is without influence on the digestibility of N, too, but the N-retention was decreased. At this leupeptin level the half-lives of intestinal proteins were prolonged. The data indicate the possibility to influence the N-retention using leupeptin.

[Isobutylene diurea as a new source of NPN for ruminants. 1. Nitrogen balance and plasma amino acid composition after feeding of isobutylene diurea to sheep]. / Isobutylidendiharnstoff als neue NPN-Quelle für Wiederkäuer

The paper first presents a survey of the existing literature where studies on the effects of isobutylidene diurea (IBDH) as NPN source for ruminants are discussed. Following this the authors discuss their own experiments on IBDH turnover. IBDH showed no toxi cation when tested in toxicity studies with rats which were carried out under standardized conditions. Within the framework of metabolism trial 4 growing male sheep (average liveweight: 30 kg) were fed a semisynthetic diet containing IBDH as sole N source. Each animal received 60 g IBDH per day. The average value for the apparent N digestibility was found to be 77.6%, the percentage for the average N ballance was 27.6%. The blood of the sheep was investigated, on a comparative basis, for the behaviour of free amino acids after the administration of an experimental semisynthetic diet containing IBDH or extracted soya bean meal as sole N source. The use of IBDH produced a higher concentration of free amino acids and a five-fold increase if urea concentrations in the blood plasma but reduced the Asp, Cys and Tyr levels when compared with the feeding of extracted soya bean meal. Similarly, IBDH supplementation increased the relative proportions of Gly, Lys, and His in blood plasma but reduced those of Asp, Val and Tyr. A positive relationship between the rate of IBDH hydrolysis and the decrease in pH was found in experiments studying the hydrolysis behaviour of IBDH at pH values from 1-7.

[Distribution of radioactivity in the body and rate of incorporation of radioactivity into the tissue proteins of monogastric animals following intravenous injection of tracer amino acids]. / Untersuchungen zur Charakterisierung der Radioaktiviätsverteilung im Organismus und zum Radioaktivitätseinbau in Gewebeproteine bei monogastrischen Tieren nach i. v.-Injektion von Traceraminosäuren

The studies were carried out with pigs and rats. The radioactive animo acids (14C leucine and 3H lysine) were administered to the pigs by way of a catheter tube into the jugular vein. Subsequently, the time pattern of the distribution of the specific amino acid radioactivity was followed in the TCE soluble and Tce precipitable fractions of the blood plasma (TCE= trichloro-acetic acid). The radioactive labelling in rats was carried out by injecting 14C leucine into the portal vein. The animals were killed after incorporation periods from 2 to 60 mins, and the levels of specific radioactivity were estimated in the TCE soluble and TCE precipitable fractions of the blood plasma, in the liver and in the skeletal muscles. The experimental results clearly indicated that the specific radioactivity of the tracer amino acids and the rate of incorporation of radioactivity into tissue proteins were greatly influenced by the size of the free amino acid pool within the range of distribution of the tracer. An estimation of the magnitude of the pool of free amino acids within the distribution range of the tracer can be obtained from the curve pattern for the decline of specific radioactivity of the corresponding free amino acid in the blood plasma. This pool exhibits a high rate of turnover. In all studies made to evaluate in vivo processes of protein synthesis by use of radioactive tracer amino acids it will be particularly important that consideration should be given to the specific radioactivity of the amino acid in the precursor pool for protein synthesis.

[The effect of proteinase inhibitors in rats. 1. Report. Effect of a short-term application on the N balance, the free amino acids in liver and muscles and the biological half-life of the intestinal tissue proteins]. / Untersuchungen zur Wirkung von Proteinaseinhibitoren bei Ratten

Short-term trials with a triazin derivative and a peptide aldehyde were carried out to investigate the in-vivo administration of proteinase inhibitors in rats for improving the N balance of the animals. The results of N balances suggest that the peptide aldehyde may be regarded as a promising substance that may be used to increase the rate of utilization of the absorbed N in the intermediary metabolism. The short-term use of both active compounds did not affect the true N digestibility. The two compounds did not influence the concentration of free amino acids in the liver and muscle tissue under the experimental feeding conditions used. Similarly, tests of the biological half-life of proteins taken from the small and large intestine did not reveal any changes due to the presence of the inhibitors. Animals receiving a protein-free diet exhibited longer half-life periods for proteins from the small and large intestine compared with animals of the other groups. A protein-free nutrition of the animals caused a general increase in the levels of free non-essential amino acids in the liver. This was accompanied by a simultaneous decrease in essential amino acids. The same type of nutrition led to a general decline in the concentration of free amino acids in muscular tissue, particularly of the essential amino acids. High-protein nutrition led especially to an increase in the concentration of branched-chain amino acids in both types of tissues investigated.

[Secretion of endogenous amino acids in the gastrointestinal tract and amino acid resorption in the swine]. / Untersuchungen zur Sezernierung endogener Aminosäuren in den Verdauungstrakt und zur Aminosäurenresorption beim Schwein

A trial was performed with 2 fistula pigs (each with 2 fistulas, one located about 30 cm below the pyloric orifice and the other at the end of the small intestine). Animal A received a casein diet containing 14% crude protein for a period of 2 weeks before the tracer amino acid was administered. Animal B received the same diet for a period of 10 days and was then fed a diet (at the same protein level) containing gluten as sole protein source. The two tracer amino acids, 14C-U-L-leucine and 3H-4,5-(N)-L-lysine, were injected intravenously. The passage rates for dry matter, organic matter and N measured at the beginning of the small intestine were higher than the rate of intake. The rate of passage of amino acids was also found to be increased relative to the rate of intake. In general, this increase involved the non-essential amino acids to a much larger extent. A considerable proportion of the amino acids passing into the large intestine is not excreted with the faeces but is probably converted in catabolic processes. It is for this reason that any values for the efficiency of amino acid absorption calculated on the basis of data on the faecal excretion of amino acids will not provide conclusive evidence for the availability of dietary amino acids in processes of the intermediate metabolism. The rate of secretion of 3H and 14C radioactivity into the digesta of the small intestine was found to increase rapidly within 1-2 hrs after administration of the tracer amino acids. The 14C radioactivity detected was found to be almost exclusively derived from 14C leucine while only about 60% of the 3H activity found in the digesta of fistula I were shown to be bound to lysine. Labelled lysine and leucine (of endogenic origin) are absorbed into the small intestine at a slower rate (i.e. endogenic proteins are less efficiently digested) than the non-radioactive amino acids (of exogenic origin) so that a process of concentration of endogenic amino acids is observed towards the end of the small intestine.