Hepatic and portal-drained viscera balances of amino acids, insulin, glucagon and gastrin in the pig after ingestion of casein or rapeseed proteins

Hepatic and intestinal balances of amino acids, insulin, glucagon and gastrin were studied in 6 non-anaesthetized Large White pigs (mean body weight 64 +/- 4.8 kg) after ingestion of casein or rapeseed proteins. The animals were fitted with permanent catheters in the portal vein, the brachiocephalic artery and the right hepatic vein. In addition, 2 electromagnetic flow probes were implanted, one around the portal vein and the other around the hepatic artery. After a preliminary adaptation to each diet the animals received at 1-wk intervals and according to a double latin square design, 3 test meals of 800 g each, one containing 23.2% of rapeseed concentrate (diet RA 12) and the others 13.9 or 27.8% of hydrochloric casein (diets CA 12 and CA 24). Each observation period lasted 12 h. Amino acids from all diets were very well absorbed. In 12 h, the absorption of total amino acids as a percentage of the ingested quantities was 99% for CA 12, 102% for CA 24 and 104% for RA 12. Hepatic uptake of total amino acids in 12 h expressed as a percentage of the absorbed quantities was 13% for CA 12, 66% for CA 24 and 25% for RA 12. Differences in the hepatic extraction rate of essential amino acids appeared between the 2 levels of casein ingestion and for Arg between the 2 protein sources. Whatever the nature of the ingested protein or the level of casein, the liver showed a net production of Asp and Glu. The production and hepatic balance of insulin were the lowest after ingestion of RA 12. No differences were noted in the same parameters for glucagon and gastrin. Independently of the nutritional situation, the hepatic extraction rate of insulin appeared to be higher than those of glucagon and gastrin. Our results showed that the nature as well as the level of dietary proteins have large effects on the sequence and volume of absorptive phenomena, the hepatic metabolism of nutrients, the production of gastrointestinal hormones and the non-hepatic tissue disposal of absorbed nutrients.     

The transformation of nitrogen and carbon in the soil during humification of straw labelled with N15

Summary Laboratory tests were made on the effect of NaNO₃ or (NH₄)₂CO₃ on the dynamics of humification in the soil of oat straw tagged with N¹⁵. The mixture was incubated for 112 days, at constant temperature and moisture conditions. It was found that NH₄-N accelerated the straw humifications more than NO₃-N. Humification started directly after the straw was introduced into the soil. N¹⁵ derived from straw consituted a part of the forming humic compounds. Already after 14 days of incubation, the N¹⁵ of straw was found in all fractions of humic compounds. Mineralization accompanied humification. The added inorganic-N accelerated not only straw humification, but also the mineralization of forming humic compounds. This is why the added inorganic-N had no influence on the content of humic compounds. The decisive factor in the increase of humic and fulvic acids in the soil, was the straw. The inorganic N added to the straw, had no influence on the quality of humic acids formed in the soil.     

Mineralization of nitrogen from15N labelled fungi,soil microbial biomass and roots and its uptake by barley plants

The effect of vesicular-arbuscular mycorrhizal fungi (VAM) on field bean and spring wheat dry matter production and on phosphorus, zinc, copper, iron and manganese uptake was determined under greenhouse conditions. Nutrient availability was varied by using different sizes of pots and by diluting the soil with sand. VAM increased plant dry matter production under all sets of growth conditions. VAM were found to directly increase the uptake of P, Zn, Cu and Fe by field beans and of P and Zn for wheat in both experiments. Increased uptake of the other nutrients measured was attributed to increased dry matter production or other factors. The effect of VAM decreased as the pot size holding the host plants decreased, but was not affected by the ratio of soil to sand if the pot size was kept constant. Nutrient uptake by beans as a proportion of total amount of nutrient present increased as the amount of nutrient decreased. Increase in root-soil contact and altered chemical equilibria are probable reasons for increased efficiency of nutrient uptake by beans as the level of available nutrient decreased. For wheat, which has a relatively fibrous root system, decreasing the nutrient availability had minimal effects on nutrient uptake in these experiments. Increases in total uptake of a particular nutrient resulting from inoculation with VAM are not necesarily indicative of a direct uptake of that nutrient by the VAM.     

Estimation of biological nitrogen fixation associated withBrachiaria andPaspalum grasses using15N labelled organic matter and fertilizer

Summary Six pasture grasses,Paspalum notatum cv batatais,P. notatum cv pensacola,Brachiaria radicans, B. ruziziensis, B. decumbens andB. humidicola, were grown in concrete cylinders (60 cm diameter) in the field for 31 months. The soil was amended with either a single addition of¹⁵N labelled organic matter or frequent small (2 kg N. ha^–1) additions of¹⁵N enriched (NH₄)₂SO₄. In the labelled fertilizer treatment soil analysis revealed that there was a very drastic change in¹⁵N enrichment in plant-available nitrogen (NO ₃ ^– +NH ₄ ⁺ ) with depth. The different grass cultivars recovered different quantities of applied labelled N, and evidence was obtained to suggest that the roots exploited the soil to different depths thus obtaining different¹⁵N enrichments in soil derived N. This invalidated the application of the isotope dilution technique to estimate the contribution of nitrogen fixation to the grass cultivars in this treatment. In the labelled organic matter treatment the¹⁵N label in the plant-available N declined at a decreasing rate during the experiment until in the last 12 months the decrease was only from 0.274 to 0.222 atom % excess. There was little change in¹⁵N enrichment of available N with depth, hence it was concluded that although the grasses recovered different quantities of labelled N, they all obtained virtually the same¹⁵N enrichment in soil derived N. Data from the final harvests of this treatment indicated thatB. humidicola andB. decumbens obtained 30 and 40% respectively of their nitrogen from N₂ fixation amounting to an input of 30 and 45 kg N.ha^–1 year^–1 respectively.     

Separation of amino acid and amide nitrogen from plant extracts for 15N analysis

A rapid method was developed to obtain nitrogen for 15N analysis of individual amino acids and amides from plant tissue extracts. Amino or amide nitrogen was recovered as ammonia, suitable for preparation of samples for 15N emission spectrometry, using a combination of ion-exchange chromatography and distillation.     

Incorporation of N from intravenously administered 15N labelled urea into the bacterial protein in the sheep.

The experiment carried out on two wethers demonstrated that nitrogen of intravenously injected urea, labelled with 15N was incorporated into total and bacterial nitrogen fraction of the digesta flowing through the rumen and duodenum. The amount of 15N in the bacterial fraction flowing throught the rumen and duodenum was relatively low in comparison with the amount of 15N in the total nitrogen (14,8% and 8,1% in the rumen and 6,6% and 7,9% in the duodenum. The ratio of the amount of bacterial-N to total-N in the rumen content (12,7 and 7,5%) was only slightly lower than the ratio of bacterial 15N to total 15N. In the duodenum this ratio was a little higher (8,7 and 10,0%). Blood urea nitrogen was utilized only partly in biosynthesis of bacterial protein. The results showed that only a small amount of blood urea nitrogen retained in the organism was utilized for microbial protein synthesis and the majority in some different way.     

Glucose absorption, hormonal release and hepatic metabolism after guar gum ingestion.

Six non-anaesthetized Large White pigs (mean body weight 59 +/- 1.7 kg) were fitted with permanent catheters in the portal vein, the brachiocephalic artery and the right hepatic vein and with electromagnetic flow probes around the portal vein and the hepatic artery. The animals were provided a basal none-fibre diet (diet A) alone or together with 6% guar gum (diet B) or 15% purified cellulose (diet C). The diets were given for 1 week and according to a replicated 3 x 3 latin-square design. On the last day of each adaptation period test meals of 800 g were given prior to blood sampling. The sampling was continued for 8 h. Guar gum strongly reduced the glucose absorption as well as the insulin, gastric inhibitory polypeptide (GIP) and insulin-like growth factor-1 (IGF-1) production. However, the reduction in peripheral blood insulin levels caused by guar gum was not associated with a change in hepatic insulin extraction. IGF-1 appeared to be strongly produced by the gut. The liver had a net uptake of the peptide. Ingestion of guar gum increased the hepatic extraction coefficient of gut produced IGF-1. Guar gum ingestion also appeared to decrease pancreatic glucagon secretion. Cellulose at the level consumed had very little effect on the parameters considered. It is suggested that the modulation of intestinal mechanisms by guar gum was sufficient to mediate the latter internal metabolic effects.     

Postprandial exchange of urea and ammonia nitrogen between the digestive tract and portal blood in the conscious pig after ingestion of a diet with or without urea

The concentrations of urea and ammonia were measured in the portal and arterial blood simultaneously to the blood flow rate in the portal vein during the postprandial period (8 hrs.) after ingestion of a normal protein diet with 3% urea (10 meals) or without urea (12 meals) in conscious pigs (mean body weight: 55.5 +/- 2.3 kg). When no urea was present in the diet, there was a slight and permanent uptake of blood urea by the gut (570 mg/h, i.e. 9,5 mmoles/h) as well as a permanent appearance of ammonia in the portal vein (258 mg/h i.e. 15.2 mmoles/h), increasing with time (P less than 0.05). The absorbed ammonia nitrogen represented a maximum of 70% of urea nitrogen taken up. 2. Addition of urea to the diet brought about a large absorption of that substance (73% of the ingested amount) followed by a rather large excretion (960 mg/h, i.e. 16 mmoles/h), 5-6 hrs. after the meal and led to an increase (P less than 0.05) in the absorption of ammonia.     

Crop uptake and leaching losses of15N labelled fertilizer nitrogen in relation to waterlogging of clay and sandy loam soils

Summary Ammonium nitrate fertilizer, labelled with¹⁵N, was applied in spring to winter wheat growing in undisturbed monoliths of clay and sandy loam soil in lysimeters; the rates of application were respectively 95 and 102 kg N ha⁻¹ in the spring of 1976 and 1975. Crops of winter wheat, oilseed rape, peas and barley grown in the following 5 or 6 years were treated with unlabelled nitrogen fertilizer at rates recommended for maximum yields. During each year of the experiments the lysimeters were divided into treatments which were either freelydrained or subjected to periods of waterlogging. Another labelled nitrogen application was made in 1980 to a separate group of lysimeters with a clay soil and a winter wheat crop to study further the uptake of nitrogen fertilizer in relation to waterlogging. In the first growing season, shoots of the winter wheat at harvest contained 46 and 58% of the fertilizer nitrogen applied to the clay and sandy loam soils respectively. In the following year the crops contained a further 1–2% of the labelled fertilizer, and after 5 and 6 years the total recoveries of labelled fertilizer in the crops were 49 and 62% on the clay and sandy loam soils respectively. In the first winter after the labelled fertilizer was applied, less than 1% of the fertilizer was lost in the drainage water, and only about 2% of the total nitrogen (mainly nitrate) in the drainage water from both soils was derived from the fertilizer. Maximum annual loss occurred the following year but the proportion of tracer nitrogen in drainage was nevertheless smaller. Leaching losses over the 5 and 6 years from the clay and sandy loam soil were respectively 1.3 and 3.9% of the original application. On both soils the percentage of labelled nitrogen to the total crop nitrogen content was greater after a period of winter waterlogging than for freely-drained treatments. This was most marked on the clay soil; evidence points to winter waterlogging promoting denitrification and the consequent loss of soil nitrogen making the crop more dependent on spring fertilizer applications.     

Uptake of 15N-labelled alanine,ammonium and nitrate in Pinus sylvestris L. ectomycorrhiza growing in forest soil treated with nitrogen,sulphur or lime

The uptake of ¹⁵N-labelled alanine, ammonium and nitrate was studied in ectomycorrhizal morphotypes of intact Pinus sylvestris seedlings. PCR-RFLP analysis of the ITS-region of fungal rDNA was used to identify the morphotypes. Seedlings were grown in forest soil collected at an experimental site in southern Sweden. The treatments compared were a control, N fertilisation (600 kg N ha^-1 as urea), sulfur application (1200 kg S ha^-1) and lime application (6000 kg CaCO₃ ha^-1). The forest, which had been dominated by Picea abies, was clear-cut two years before the forest soil was sampled. Soil was also collected from an adjacent standing forest. The aim of the present study was to detect changes in the ectomycorrhizal communities in forest soils and relate these changes to the functional parameter of uptake of nitrogen from organic (alanine and protein) and inorganic (ammonium and nitrate) sources.Liming resulted in the detection of a morphotype not found in other samples, and one morphotype was only found in samples from the standing forest (the fungi in these two morphotypes could not be identified). All mycorrhizal root tips showed a higher ¹⁵N concentration after exposure to different nitrogen forms than non-mycorrhizal long roots. Uptake of¹⁵ N from a labelled solution of alanine or ammonium was higher (about tenfold) than uptake from a ¹⁵N-labelled solution of nitrate. Uptake of ammonium and alanine varied between 0.2 and 0.5 mg N g^-1 h^-1 and between 0.1 and 0.33 mg N g^-1 h^-1, respectively, among the different morphotypes.In seedlings grown in the control soil and in soil from standing forest, alanine and ammonium were taken up to a similar degree from a supply solution by all morphotypes, whereas ammonium uptake was higher than alanine uptake in seedlings grown in lime-treated soil (about twofold) and, to a lesser extent, in the nitrogen- and sulfur-treated soils. The higher ammonium uptake by morphotypes from the limed soil was confirmed in pure culture studies. In cases where ammonium was used as the N source, an isolate of the S. variegatus morphotype collected in the limed soil produced more biomass compared with isolates of S. variegatus collected in nitrogen- or sulphur-treated soil. One isolate of a silvery white morphotype produced about equal amounts of biomass on alanine and ammonium, whereas all S. variegatus isolated performed better with ammonium as their N source. Based on the results it is hypothesised that liming can induce a shift in the ectomycorrhizal community, favouring individuals that mainly utilise inorganic nitrogen over those that primarily utilise organic nitrogen.     

Proteinogenic amino acids labelled with 15N and/or 13C for application in peptide synthesis: A short review with a comprehensive list of published derivatives

A review is given of the literature dealing with the most common protected derivatives of ¹⁵N- and/or ¹³C-labelled amino acids of interest in peptide synthesis. The list contains all such Boc-, Z- and Fmoc-amino acids as well as published methyl, ethyl, t-butyl and benzyl esters.     

Biosynthesis of the glucobrassicin aglycone from14C and15N labelled L-tryptophan precursors

The formation of the isothiocyanate group of the aglycone of glucobrassicin (Glubr) an indole glucosinolate present in relatively large quantities in plants of the familyBrassicaceae, was studied. Labelled Glubr was synthesized from L-tryptophan-3-¹⁴C-amino¹⁵N in winter rape hypocotyl segments. After extraction it was finally isolated in a crystalline state by mixed crystallization with added inactive Glubr tetramethylammonium salt. The specific activity of¹⁴C and the atoms % excess¹⁵N were determined in both the precursor and the product. The ratio¹⁴C/¹⁵N as well as the values of dilution of¹⁴C and¹⁵N were in agreement in the precursor and in the final product. This shows,inter alie, that during the biosynthesis of Glubr, L-tryptophan is not deaminated. Thus the presumed isothiocyanate group of Glubr is synthesized on the α-carbon of the L-tryptophan alanine chain, the α-carbon together with the attached nitrogen pass directly from the precursor into the Glubr aglycone. This represents a type of glucosinolate aglycone biosynthesis where the carbon chain of the initial amino acid is not elongated. This finding is supported by the fact that L-tryptophan-l-¹⁴C does not yield labelled Glubr the C₁ carbon being decarboxylated during Glubr synthesis.     

In vivo 15N NMR studies of regulation of nitrogen assimilation and amino acid production by Brevibacterium lactofermentum

Glutamic acid producer Brevibacterium lactofermentum intact cells were used to demonstrate the feasibility of in vivo 15N NMR to follow nitrogen assimilation and amino acid production throughout the growth cycle. The induction of glutamic acid production by different growth conditions was studied. Intracellular and extracellular levels of free metabolites were estimated as function of oxygen supply and biotin concentration. 15N NMR enabled us to distinguish two phases during the fermentation. At the early stage of fermentation, glutamic acid was accumulated intracellularly independent of oxygen supply and no product was excreted. In the late growth phase, the permeability of the cells developed and L-glutamic acid was excreted. The effect of aeration and biotin concentration on cellular contents and excretion was also studied by 15N NMR. Glutamate, N-acetylglutamine, and glutamine were the main nitrogenous pools independent of cell culture conditions. Free ammonia was not accumulated intracellularly although glutamic acid fermentation can be characterized as the process of nitrogen assimilation and the uptake of ammonia is the key step. In conclusion, the application of in vivo 15N NMR spectroscopy unraveled various problems of nitrogen metabolism, in a rapid and nondestructive manner.     

Relative concentrations of N15 in urinary ammonia N and urea N after feeding N15-labeled compounds

Available data on the isotopic ratio See PDF for Equation of ammonia (r_a) and that of urea (r_u) after a single feeding of glycine, aspartic acid, and ammonium citrate are analyzed. From this analysis the following conclusions are drawn. 1. The isotopic ratio See PDF for Equation of ammonia (r_a) is always higher than that of urea (r_u) in the initial period after a single feeding of isotopic glycine or aspartic acid, but the relation is reversed later. A similar relation probably holds after feeding isotopic ammonia. 2. It is pointed out that the ratio of average r_a to average r_u depends on the time interval for which urine is collected, on the schedule of feeding, and probably also on the amount taken at each feeding. When the amount fed and the feeding schedule are unknown, theoretical interpretation of the ratio of average r_u to average r_u is impossible. 3. At the point of maximum isotopic ratio of urea, it is very probably equal to the isotopic ratio of ammonia. A possible explanation is suggested.     

Effects of ileal endogenous nitrogen losses and dietary amino acid supplementation on nitrogen retention in growing pigs

The relationship between ileal endogenous nitrogen (N) losses and N retention was studied in two experiments with growing pigs of 40 to 60 kg. In Experiment 1, 13 ileal cannulated castrated males were fed diets based on maize starch, containing either soyabean meal (SBM) with a low trypsin inhibitor activity (TIA), a mixture of toasted and untoasted soyabean meal with a high TIA (mSBM), a commercial batch of peas, or rapeseed expeller cake (RC). Ileal endogenous N recovery was measured using the ¹⁵N-isotope dilution technique. Apparent ileal digestibility of crude protein (CP) for the SBM, mSBM, pea and RC diets were 82.8, 72.0, 76.7 and 68.7% (P < 0.05). True ileal CP digestibility for the diets was 96.5, 93.0, 94.0 and 87.5% (P < 0.05), and the recovery of ileal endogenous N was 3.08, 6.01, 4.55 and 5.36 g/kg DMI (P < 0.05). In Experiment 2, sixteen castrated males were used to determine N retention, using almost similar diets as in Experiment 1. The diets contained either SBM, mSBM or peas and were balanced for the contents of apparent ileal digestible (ID) CP (96 g/kg) and ID essential amino acids (EAA; at least 85% of requirement values). The fourth treatment was a diet with mSBM as protein source, but supplemented with EAA to the level of 95% of the requirement values (diet mSBMs). Apparent faecal CP digestibility for the SBM, mSBM, pea and mSBMs diets was 88.6, 87.2, 86.1 and 86.0% (P < 0.05). Urinary N excretion and N retention for these treatments were 0.39, 0.59, 0.40, 0.53 (P < 0.05) and 0.87, 0.80, 0.85, 0.84 g/kg^0.75/day (P < 0.05), respectively. Utilization of dietary ID N for N retention were 79.8, 73.3, 78.2 and 77.6% (P < 0.05), respectively. The study showed that increased ileal endogenous N losses are associated with higher losses of urinary N and with a lower N retention. Supplementation of extra essential AA to a diet causing a relatively high flow of ileal endogenous N, may compensate for the lower N utilization under these conditions, and thus limit effects on N retention.