Effect of glutamic acid content of the diet on the catabolic rate of isotope-labeled glutamic acid in rats. 2. Time course of 14CO2 excretion following subcutaneous administration of 14C-glutamic acid

40 rats with a body weight of 100 g received 7 semisynthetic diets with different contents of glutamic acid and one diet contained whole egg. A L-amino acid mixture corresponding to the pattern of egg protein was the protein source of the semisynthetic diets. Glutamic acid was supplemented successively from 0 to 58 mol-% of the total amino acid content. On the 8th day of experimental feeding the animals were labelled by subcutaneous injection of 14C-U-L-glutamic acid. Subsequently the CO2- and the 14CO2-excretion were measured for 24 hours. In this period 64 to 68% of the injected radioactivity were recovered as 14CO2. The curve pattern of 14CO2-excretion indicates two different processes of 14CO2-formation. One characterizing the direct degradation of glutamic acid to CO2 with a high rate constant and a second one with a lower rate constant characterizing the 14CO2-formation via metabolites of glutamic acid. 77% of the total 14CO2-excretion in 24 hours resulted from the direct oxidation of glutamic acid and 23% from the oxidation of intermediates. When 14CO2-formation was measured 10 to 24 hours after injection of 14C-glutamic acid a positive correlation to the content of glutamic acid in the diet was observed. The intestinal tissue contribute considerably to the catabolization of glutamic acid, however, there seems to exist an upper limit for this capacity.     

Effect of glutamic acid content of the diet on isotope-labeled glutamic acid catabolism in rats. 1. The course of 14CO excretion following intragastric administration of 14C-glutamic acid

Male rats received in 8 groups of 10 animals each for a period of 7 days 7 synthetic diets and one semisynthetic diet on maintenance requirement level. A L-amino acid mixture corresponding to the pattern of egg protein without glutamic acid was the protein source of the synthetic diets. Glutamic acid was supplemented successively from 0 to 58 mol-% of the total amino acid content. The crude protein source of diet 8 was whole egg powder. On the 8th day of experiment 5 animals per group were labelled by intragastric infusion (i.g.) with 14C-U-glutamic acid. During the following 24 hours the excretion of CO2 and 14CO2 was measured. Throughout the experimental feeding body weight was relative constant, however, when the synthetic diets were fed it was necessary to increase the daily amount of energy from 460 to 480 kJ/kg0,67. The relative 14CO2-excretion within 24 hours was 68-75% of the dose. However, the main part of the amount of radioactivity excreted during 24 hours was found after 4 to 6 hours already. Exponential functions calculated from the data of cumulative 14CO2-excretion suggest the existence of a fast process of 14CO2-formation directly from 14C-glutamic acid, reaching a plateau within 2 hours and a slow process of oxidation of intermediates of glutamic acid metabolism, causing a continued 14CO2-formation even after 24 hours. The oxidation of 14C-glutamic acid to CO2 decreased 2 to 14 hours after i.g. labelling if the glutamic acid content of the diet increased. The same was found for the specific radioactivity of 14CO2. A storage of intermediates of glutamic acid before degradation was assumed.     

Effect of glutamic acid content in the diet on the catabolic rate of the isotope-labeled glutamic acid in rats. 4. Measurement of 14C-glutamic acid oxidation to 14CO2 with various energy sources in the diet

48 male rats (body weight 80-100 g) were fed with 2 diets different in the glutamic acid content (diet I 2.42 and diet II 6.24% glutamic acid in the dry matter). The mixture of the other synthetic L-amino acids was adapted to the egg protein pattern corresponding 10% crude protein in the diet. Each diet was fed either on 73% or 98 to 104% of the energy maintenance requirement. After 7 days of experimental feeding 14C-U-L-glutamic acid was given to each group by intragastric infusion (i.g.), intraperitoneal (i.p.) or subcutaneous injection (s.c.), respectively, followed by a measurement of the CO2-and 14CO2-excretion during two subsequent periods of 3 hours. The CO2-excretion was lower in animals with restricted energy intake especially during the first 3 hour-period, which was started 2 hours after feed intake. The relative 14CO2-excretion (percent of the dose) was neither significantly influenced by the level of energy intake nor by the amount of dietary glutamic acid. The highest degradation rates of 14C-glutamic acid to 14CO2 were measured after i.g. application (more than 50%), followed by the i.p. injection (nearly 50%) and the lowest values were observed after s.c. injection (about 40%). These differences were only evident during the first CO2-absorption period. Furthermore the s.c. injection caused a lower specific radioactivity of CO2 compared with the data after i.g. and i.p. application. The results suggest the high metabolic activity of the intestinal tissue for glutamic acid.     

Studies on the 14C-15N-acetamide turnover in sheep. 2. Studies on 15N turnover and comparative studies on the 14C turnover]

Three fistula sheep with average weights of 52.2 kgs were given 37.9 g of 15N and 14C labelled acetamide (= 1.09 mg 15N' and 0,95 mCi14C) which were administered directly through the fistula. The half-life period of 15N retention in the ruminal fluid (TCE soluble portion) was found to be 4 hrs. 18 hrs after 15N administration increasing amounts of 15N were carried back to the rumen by way of the rumino-hepatic circulation. The 15N concentration in the blood (TCE soluble portion) rapidly increased up to a peak value and, from 3 hrs after isotope administration, the 15N concentration was found to decline continuously, with a slight discontinuation at about the 10th hr of experiment. The rate of 15N incorporation into the protein fraction (TEC soluble portion) of the blood was delayed by 4 hrs, relative to the rate of 15N incorporation into ruminal proteins. An average of 43.1% of the administered amount of 15N was excreted in the urine within 7 days. Up to the 4th day of experiment the half-life period of urinary 15N excretion was 19 hrs. An average of 15% of the administered total amount of 15N was excreted in the faeces. In this process, the peak values in both TCE fractions were observed to occur on the 2nd day of experiment. The proportion of isotope in the TCE soluble fraction was found to increase continuously compared with the total amount of the isotope excreted in the faeces. Isotope concentrations between 0.03 and 0.13 atom% of surplus 15N were found in organ and muscle tissues of a sheep that had been slaughtered 7 days after administration of the isotope. The results obtained are discussed on the basis of comparisons made with the analogous behaviour of 14C activity.     

Endogenous nitrogen metabolism in 15N-labeled swine. 3. Endogenous excretion of 15N- and 14C-labeled secretions following a 14C-leucine injection

Four pigs (59-65 kg live weight) were labelled over a period of 10 days with 15N in the feeding of a fishmeal diet (1), a fishmeal diet + partly hydrolysed straw meal (2), a horse bean diet (3) and a horse bean diet + partly hydrolysed straw meal (4). After a 24-hour fasting the animals were provided with simple cannulae in the upper part of the small intestines. After a fasting period of 24 h all four pigs received a 14C-leucine injection and the cannula secretion was collected in the subsequent 24 h. After the feeding of the diets without straw meal supplement (1 and 3) there were distinct differences in the secretion in comparison with the feeding with straw meal supplements (2 and 4) despite the long fasting period (48-72 h). 14C-activity could already be detected in the TCA-precipitable fraction of the secretion after 3-6 min of the injection in 1 and 3 but only 20 to 25 min after the 14C-leucine injection in 2 and 4. The specific 14C-leucine activity of the TCA-soluble fraction of the secretion was, after the straw meal supplementation to the fish meal diet, 15 times higher 25 min after the 14C-leu-injection, 25 times higher after 70 min, 36 times after 2 h and 1.8 times after 4 h than without straw meal supplementation. For all four diets a specific correlation (r = 0.96) could be ascertained between the increase of 14C-activity/mg N in the TCA-soluble fraction and the increasing crude fibre content in the diet between 25 and 180 min after the injection. Furthermore, a distinctly decreased N-secretion/h could be ascertained (correlation coefficient r = 0.84) with the increasing crude fibre content in the diet. The influence of the crude fibre on the parameters mentioned is seen in the changed osmotic conditions in the secretion, which may be caused by the changed regulation by hormones of the gastro-intestinal tract. The atom-% 15N' in both TCA-fractions of the secretion underwent big rhythmic variations, which is explained by different ratios of the components pancreatic juice, bile, and intestinal juice.     

Determination of lysine requirement in growing rats as based on the catabolism rate of 14-C- and 15-N-labeled lysine

Male Wistar rats (weighing some 80 g at the start of the experiment) were fed diets containing maize gluten as protein carrier and which was supplemented with amino acids (except lysine) in such way that their concentrations came up to the requirement norms. Lysine was gradually supplemented this resulting in 10 diets of different lysine content (1.6-10.6 g lysine/16 g N). On the 7th experimental day, 4 animals of each group were labelled with 14C-lysine and subjected to 2-hour measuring of 14CO2-excretion. On the following day, the animals were injected i.p. 15N-lysine, the urine being collected over 24 hours to determine 15N-frequency in urine. Both 14CO2-excretion and 15N-frequency in urine were found to remain constant at a lysine content of the diet up to 4.5 g/16 g N and rose steeply from 5.8 g lysine/16 N on. Under the experimental conditions chosen the lysine requirement is deduced to be 5 g/16 g N. This method of lysine requirement determination is highly sensitive and exact because it covers the catabolization of the amino acids under study and not so parameters that are known to be influenced by other factors such as growth, N-balance, total N-conversion or CO2-formation. The method can also be applied to metabolic situations not connected with productive performances.     

Dynamics of amino acid and protein metabolism in laying hens after the administration of 15N-labeled wheat protein. 2. 15N excretion with nitrogen and basic amino acids of the feces

Over 4 days 12 colostomized laying hens received together with a commercial ration labelled wheat with a 15N excess (15N') of 14.37 atom-%. The labelling of the basic amino acids amounted to 13.58 atom-% for lysine, to 14.38 atom-% for histidine and to 13.63 atom-% 15N' for arginine. 3 animals each were butchered 12 h, 36 h, 60 h and 108 h resp. after the last application of 15N. The heavy nitrogen in the total N and in the N fraction of non-protein origin as well as in the basic amino acids in faeces was daily determined for the individual hens in the total experimental period. On average the crude protein of faeces contained 5.45 % lysine, 2.32% histidine histidine and 3.68% arginine: the protein of faeces correspondingly contained 5.43% lysine, 2.32% histidine and 4.07% arginine. The quota of TCA soluble N in the total N of faeces amounts to one third on the 3rd und 4th days of the experiment and that of 15N' to 28%. The average atom-% 15N' of the protein fraction is 3.48 atom-% 15N' and that of the non-protein N fraction of faeces 2.93 atom-% 15N'. The apparent digestibility and that of the non-protein N fraction of faeces 2.93 atom-% 15N'. The apparent digestibility of the 14N of the ration on average amounts to 82.8% and that of the wheat 15N' to 87.5%. The average quota of the basic amino acids in the protein compounds of faeces amounts to 70.9% for lysine 15N', 73.7% for histidine 15N' and 70.3% for arginine 15N'. The digestibility of the 15N labelled amino acids amounts to 80.4% for lysine, 90.8% for histidine and 90.2% for arginine.     

Methodologic studies on the metabolically-oriented determination of lysine requirements in broiler chickens. 2. Effect of the lysine content of the diet on the lysine oxidation rate in a 15-hour feed withdrawal before 14C-lysine injection

This experiment was designed to narrow the estimated range for lysine requirement of broiler chickens determined by isotopic techniques. In addition the influence of a long-term feed withdrawal previous 14C-lysine-injection on the lysine catabolism was investigated. 120 male broiler chickens 7 to 21 days posthatching received a diet based on wheat and wheat gluten. Lysine content was varied from 8.3 to 16.0 g/kg DM (3.2 to 6.3 g/16 g N) at 8 levels by supplementing the basal diet with L-lysine-HCl. After the feeding period animals of each group were labelled with 14C-L-lysine by intravenous injection 5.5 and 15.5 hours after feed withdrawal, respectively. During the following 4 hours the excretion of 14CO2 and CO2 was measured. Highest body weight gain was observed in the group with 13.8 g lysine/kg DM. In case of 14CO2 excretion measurements starting 5.5 hours after feed withdrawal an increase of 14CO2 excretion was observed if the lysine content of the diet exceeded 11.6 g/kg DM. This estimated range for lysine requirement (11.6 to 12.7 g/kg DM with 26% CP in the DM) was lower compared with the lysine requirement estimated by the growth curve (12.7 to 13.8 g/kg DM). This discrepancy could be explained by the fact that the results of the metabolism oriented determination of lysine requirement represent the requirement at the actual age, while the feeding experiment reflects a mean lysine requirement of the previous period of 14 days. If the animals were labelled with 14C-lysine 15.5 hours after feed withdrawal no clear response in 14CO2 excretion and specific radioactivity of CO2 on the dietary lysine content was observed.     

Volatile compound content and fatty acid composition of pork as influenced by linoleic acid content of the diet.

Eighty pigs (average weight of 60 kg) were allotted by weight and sex to pens and treatments. There were four dietary treatments, five pens per treatment, and four pigs per pen. Diets consisted of a typical corn-soybean mix containing 9% total fat, 3% from the corn-soybean mix and 6% added. The four dietary treatments included 1) 6% safflower oil, 2) 4% safflower oil and 2% tallow, 3) 2% safflower oil and 4% tallow, and 4) 6% tallow, resulting in 6.1, 4.6, 3.2, and 1.76% linoleic acid, respectively, in the diet. Pigs were slaughtered at an average weight of 100 kg. Proximate composition, tristimulus color coordinates (L, a, and b values), pH, and flavor difference of the longissimus muscle (LM) were evaluated. Fatty acid content (milligrams per 100 grams of tissue) of the subcutaneous fat and LM and headspace volatile content of the LM were determined by capillary gas liquid chromatography. Proximate composition, color, pH, and flavor of the LM were not influenced by diet. Fatty acid content of the subcutaneous fat and LM and volatile content of the LM were influenced by diet. Increased levels of safflower oil in the diet resulted in less C16:0 and C18:1 and more C18:2, C20:2, and C20:3 in the subcutaneous fat. The LM contained more C18:2 and less C18:3 and C24:0 due to increased levels of safflower oil in the diet. Compared with the 6% tallow diet, LM from pigs fed the 4 or 6% safflower diets contained more pentanal, hexanal, 2-heptanone, trans-2-heptenal, 2-pentyl furan, 2-ethyl-1-hexanol, decanal, and undecanal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Utilization of N15-labelled urea in the laying hen. 5. N15-labelling of the content and tissue of separate parts of the gastrointestinal tract

In the series of experiments with labelled urea three colostomized laying hybrids were butchered after a six-day application of 1% urea with 96.06 atom-% 15N excess (15N') in the ration and another 2 days with a supplement of 1% unlabelled urea. Out of the individual samples from crop, gizzard, small intestine, caecum and rectum, the content of the small intestine and the caecum showed the highest labelling with greater than 1 atom-% 15N'. The TCA soluble fraction of the content of the gizzard was more highly and that of the intestines less labelled than the total nitrogen. The tissue of the gizzard is distinctly less labelled than the "omasum system" and the small intestine. The atom-% 15N' of the oesophagus with crop and glandular stomach largely showed agreement in the individual hens with that of intestinal tissue and ranged between 0.71 and 0.89 atom-%. 2% of the 15N' supplemented with the urea could be recovered in the content and the tissue of the gastro-intestinal tract.     

Studies on methods for the determination of amino acid requirements for metabolic balance based on the oxidation rate of 14C labeled lysine to 14CO2]

Male experimental rats (100 gm liveweight) were distributed into 10 groups of 8 animals each and received balanced diets, with the exception of lysine which was added to the diets in graded amounts in such a way that the lysine content of the diets ranged from 2.44 to 5.92 gm/16 gm N. After a feeding period of 7 days the animals received 3H- and 14C lysine injected intraperitoneally, 4 animals of each group were investigated for the total CO2 excretion and 14CO2 excretion during the first 2 hrs after the injection and for the urinary excretion of radioactivity (48 hours). The remaining animals in each group were used for determining the plasma amino acids and for establishing the specific radioactivity of free lysine in the liver and muscles after an 1-hour incorporation period. Total CO2 excretion was not found to be influenced by the lysine contents while the level of excretion of 14C activity through CO2 and that of specific 14C activity of CO2 increased with increasing lysine concentrations. This produced a broken curve pattern, showing an increased release of 14CO2 (under maintenance conditions) if the diet contained 4 gm lysine/16 gm N and more. Investigations for the specific 14C activity of free lysine in the liver, the main site of lysine oxidation, showed that the increase in 14CO2 release was due to an enhanced rate of lysine catabolism and was not brought about by changes in the pool volume or in specific radioactivity. The levels of urinary 14C excretion were not found to be related to the lysine content of the diets, whereas the curve pattern of 3H excretion observed 5 to 8 hrs after injection was similar to that of 14CO2 excretion. The lysine content of blood plasma and the content of free lysine in the liver increased continuously with increasing levels of dietary lysine. The methodological studies made in the present paper showed that in scientific research a determination of amino acid requirements on the basis of CO2 oxidation data may be a very exact and sensitive method. It will also yield values for maintenance requirements.     

Endogenous nitrogen metabolism processes in 15N-labeled swine. 2. Fecal excretion of amino acids and 15N-labeled amino acids with diets differing in crude fiber content

Four pigs were labelled with 15N-ammonium salt over a period of 10 days in the feeding of a fishmeal diet, a fishmeal diet + partly hydrolysed straw meal, a field bean diet and a field bean diet + partly hydrolysed straw meal. The 14N-amino acids and the 15N-amino acids excreted in faeces showed highly significant correlation coefficients with the increasing content of crude fibre in the diets, which amounted to 3.0, 5.3, 10.0 and 12.1% in the DM. The following sequence was established for the growth angle (tan alpha) of the essential 14N-amino acids: Leu, Lys, Arg, Thr, Phe, Ile, Val, His and of the 15N-amino acids: Lys, Arg, Val, Leu, Ile, Thr, Phe and His. As Lys, His and Thr cannot incorporate 15N in transamination reactions in the intermediate metabolism, their level of labelling was considerable in case of diet 4. Nevertheless, tan alpha is highest for 15N-Lys and lowest for 15N-His. This means that His in contrast to Lys, parallel to increased synthesis, is also increasingly decomposed in the large intestine. In contrast to this, proline was not labelled with 15N even with the highest content of crude fibre in the diet. Despite this, 14N-proline excretion, next to glutamic acid, increased most with the growing content of crude fibre in the diet. Due to the hydrophilic character of glutamic acid and the increased water influx in the large intestine and the increased content of crude fibre in the diet, a growing proline transport parallel to the increased influx of crude fibre and water must be assumed. If the growth angle tan alpha for the excretion of 14N-amino acids is ascertained regressively for a crude fibre content of diet of 10%, one can prove from the proportion of the amino acids and a comparison from literature for faecal bacteria and ileum digesta that the amino acid composition for this measuring point largely corresponds to that of bacteria protein.     

Methodological aspects of the determination of nitrogen metabolism parameters from 15N-tracer experiments on swine based on nitrogen metabolism models. 2. Calculation of N metabolism parameters from the time of urinary excretion of 15N

After a 45-hour infusion of 15N-lysine and 15N-glycine resp. with pigs of 25 kg live weight, the infusion of the tracer was disrupted and a 3-day reduction phase was made part of the experiment. The N-metabolism parameters such as protein synthesis and decomposition quotas were calculated on the basis of the temporal development of 15N-amounts excreted in urine. The labelling and reduction phases were evaluated simultaneously. By means of including the total N-amount of the animal body the balance calculation becomes more stable. The possible influence of the tracer amino acid in connection with the 3-pool-model is discussed on the basis of the ascertained parameters.     

Utilization of 15N marked urea by the laying hen. 2. Incorporation and metabolism of 15N for the synthesis of egg protein

In an N-metabolism experiment 3 colostomized laying hybrids received 2870 mg 15N-excess (15N') per animal in 6 days in the form of urea with their conventional feed rations. During the 8-day experiment the 21 eggs laid were separated into eggshell, white of egg and yolk. Weight, N-content and 15N' were determined of the individual fractions of the eggs. On an average of the 21 eggs 4.6% of the heavy nitrogen was in the egg-shells, 50% in the white of egg and 45.5% in the yolk. 2.8%, 4.5% and 5.5% (hens 1...3) of the 15N' consumed were detected in the eggs. The maximum 15N'-output in the white of egg was reached on the 6th day, whereas 15N'-output in the yolk showed a nearly linear increase in the time of the experiment. The results show that labelled nitrogen from urea is incorporated into the egg to a lower degree than after the feeding of 15N-labelled proteins and that the development of its incorporation into the white of egg and the yolk differ from that after the feeding of 15N-labelled native proteins.     

Effect of a supplement of partly hydrolized straw meal to a diet of fish meal and horse beans on N balance and amino acid excretion in the feces of swine

2 test pigs each (male castrated pigs) of an average live weight of 51-71 kg received either a wheat/fish meal diet (Ia) or a wheat/horse bean diet (IIa). Due to a supplement of partly hydrolysed straw meal the crude fibre content was increased from 3.0% (= Ia) to 10.1% (= Ib) and from 5.2% (= IIa) to 12.1% (= IIb) in the dry matter. This straw meal supplement decreased the apparent digestibility of the crude protein in I from 92.5 to 85.7% and in II from 89.0 to 79.0%. N-excretion in faeces/100 g DM-intake followed the regression line: y = 228 + 31.2x(r = 0.98) y = mg N/100 g DM-intake x = % crude fibre in the DM of the diet The excretion in faeces/100 g DM-intake of 15 investigated amino acids, too, in each case showed a significant increase with the rising crude fibre content of the diet. The widest growth angle (tan alpha) of all the essential amino acids investigated was shown by lysine and those amino acids with branched chains. The conclusion is that in the presence of a fermentable crude fibre source and a good N-supply those 4 amino acids show a suitable bacterial synthesis rate and a low decomposition rate.     

Resorption and incorporation of radioactive-labeled amino acids during the administration of various protein carriers in rats. 2. Uptake of radioactivity by tissues of the gastrointestinal tract after intragastric administration of 14C-L-U-leucine and 3H-glycine]

Male Albino rats (weighing 90-100 gms) were fed ad libitum for 14 days with limited periods of access to food. Powdered whole egg (V), fish meal (F), yeats (H), and gelatine (10% protein in dry matter) used as protein sources. Additionally, one group of rats received a protein-free (e). Radioactive tracers were administered by intragastric infusion of 25 mu Ci 3H glycine and 5 mu Ci 14C L-leucine per 100 gm of body weight 2 hrs after the feeding of 2 gm of the experimental diet per 100 gms of body weight. The level of uptake of radioactive tracers from the different sections of the gastro-intestinal tract was measurels of 3H and 14C labelling in intestinal tissues were observed 3 hrs and 7 hrs after infusion. The level of 14C labelling was found to be negatively correlated and the level of 3H labelling was positively correlated with the biological value of the diet. Intestinal tissues are capable of storing considerable amounts of 14C radioactivity. So, 72 hrs after infusion, the following levels of 14C radioactivity (expressed as percentage of the total dose of radioactivity) were found in tissues of the gastro-intestinal tract: whole egg: 8.4%, fish meal: 9.6%, yeast: 13.1%, gelatine: 14.9%; protein-free diet; 14.2%. The quotients correlating the levels of radioactivity from the intestinal contents with that found in the intestinal wall suggest that the walls of the small intestine possess a high capacity for absorption. At all times of radioactive measurements the walls of the small intestine were found to contain higher levels of both 14C and 3H radioactivity than the contents of small intestine.     

Effect of dietary corn oil supplementation on equine gastric fluid acid, sodium, and prostaglandin E2 content before and during pentagastrin infusion

The effect of corn oil (approximately 60% [wt/vol] linoleic acid) dietary supplementation on various components of equine gastric secretion was studied by use of a repeated-measures experimental design. Four healthy adult ponies were surgically fitted with gastric cannulas. The ponies were then fed a free-choice hay diet for 5 weeks, which was followed by 5 weeks of the same diet supplemented with 45 mL of corn oil daily. Gastric contents were analyzed under basal and pentagastrin-stimulated conditions once weekly during the latter 2 weeks on each diet. Gastric contents were collected at 30-minute intervals, and volume, hydrogen ion concentration, sodium content, and prostaglandin E2 (PGE2) content were measured. Data were analyzed by a linear fixed-effect modeling procedure. During the diet supplemented with corn oil, the ponies had, under basal and pentagastrin-stimulated conditions, significantly decreased acid output and significantly increased PGE2 and sodium outputs compared to those measured before corn oil supplementation. We conclude that corn oil supplementation may be an effective and inexpensive way to increase the protective properties of equine glandular gastric mucosa. This could be particularly helpful in reducing the chances of ulceration associated with nonsteroidal anti-inflammatory drug (NSAID) administration.     

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

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.     

Resorption and incorporation of radioactive labeled amino acids during administration of various protein carriers in rats. 1. Resorption of 14C leucine and 3H glycine after intragastric administration]

Male Albino rats (90-100 g) were fed ad libitum (with limited periods of feeding) for 14 days. The diets were adjusted to a crude protein content of 10%. Powdered whole egg, fish meal, yeast and gelatine were used as protein sources. Additionally, one group of rats was fed a protein-free diet. On the 15th day of experiment the rats were fed a test diet at a level of 2 g per 100 g of body weight. 2 hrs after that the rats received 25 muCi of 3H glycine and 5 muCi of 14C-L-Leucine per 100 g of body weight administered by way of intragastric infusion. It was found that a large proportion of the radioactive amino acids were absorbed as early as after 0.5 hr. The highest rate of absorption was observed in animals fed dietary proteins of poor quality or a protein free diet, so that in animals receiving a gelatine diet or a protein-free diet only 68.4% or 56.4% of the administered amount of 14C activity were detected inside the gastro intestinal tract after 0.5 hr. Analogous data for the 3H activity were 52.4% and 25.3%. Maximum absorption occurred after 3-7 hrs. Following this the level of radioactivity in the intestinal contents again increased reaching a peak value after 14-24 hrs; in the case of 14C activity this peak value amounted to 25.4% of the administered dose in animals fed the gelatine diet and 32.8% in the group receiving the protein-free diet. It was established that the major proportion of the resecreted amount of 14C activity was present in leucine. Until 72 hrs after the intake of 14C activity the level of radioactivity was again found to decline, a processes which was induced by processes occurring in the large intestines. Moreover, evidence was obtained in confirmation of previous findings, indicating that the composition of faecal amino acids was constant and unaffected by dietary proteins.