Studies of the protein and the energy metabolism in man during a wintering in Antarctica

During the 29th Soviet Antarctic Expedition in Novolazarevskaya from March 1984 to March 1985, the protein and energy metabolisms were studied in six expeditioners from the German Democratic Republic. The investigations were carried out at the beginning of the expedition (May), during the polar night (July) and during the polar day (December). The effect of a special stress situation (sledge trek in April 1984) was investigated in one subject. The stable nitrogen isotope (15)N was used to study the protein metabolism. The assessment of the energy metabolism was based on the oxygen consumption, which was determined by means of a spirograph. In addition, the vital capacity, the breath minute volume, the blood pressure, etc. were measured. The following results were obtained: During the polar night, the utilisation of the dietary proteins and the whole body protein synthesis calculated by means of the (15)N excretion of the total nitrogen in urine were greater (73.6±0.9 % and 3.48±0.17?g protein d(-1)?kg(-1), n=3) than the respective values during the polar day (69.7±1.2, p<0.05, n=3 and 3.05±0.07, p<0.05, n=3) and at the beginning of the expedition (69.6±1.4, p<0.02, n=5 and 2.81±0.09, p<0.01, n=5). The lowest values (58.0 % and 2.43?g protein d(-1)?kg(-1)) were obtained in the subject after the trek. The resting metabolic rate (in kJ?d(-1)?m(-2)) was decreased during the polar night (45.6±5.0, n=4) in comparison with the polar day (61.5±11.3, n=3) and the beginning of the expedition (52.3±9.6, n=4) with p<0.01 in both cases.     

Theoretische Aspekte der Untersuchung des Stickstoffmetabolismus mit 15N beim Menschen II. Probleme der Auswertung und Interpretation klinischer Untersuchungen

Klinische Untersuchungen zum menschlichen Stickstoffmetabolismus, die unter Verwendung des stabilen Stickstoffisotops ¹⁵N durchgeführt wurden, werden mit Hilfe eines 3-Compartmentmodells ausgewertet und interpretiert. Möglichkeiten zur Erweiterung dieses Modells werden diskutiert, und durch Computerstudien wird die Aussagekraft der aus Modellen gewonnenen Resultate überprüft.     

Assessing the subsequent effect of nitrogen released from tobacco-waste on maize crop using a 15N isotope technique

The investigation of the residual effect of nitrogen (N) released from tobacco-waste (TW) using isotope techniques will provide valuable data for sustainable organic farming. For this aim, a pot experiment was conducted using the ¹⁵N isotope technique. The experiment was based on a completely randomised design with four replications and was conducted on a calcareous ustochrepts soil. TW at levels of 0, 10, 20, 30 and 40 t ha^?1 and N fertiliser as (NH₄)₂SO₄ at levels of 0, 20, 40, 60 and 80 kg N ha^?1 were used for the Bezostaja-1 wheat variety. Concerning mineral N fertilisation with 20 and 80 kg N ha^?1, additional treatments with ¹⁵N-labelled (NH₄)₂SO₂ (10 at.% exc.) have been applied. Following harvesting wheat plants, the Pioneer 3377 maize variety was used to see the residual effect of TW. After harvesting, dry matter yields were recorded and total N concentrations were determined. ¹⁵N determinations and calculations were also made for ¹⁵N treatments separately. TW had a significant residual effect on the growth of corn plant under the pot condition. Increasing rates of TW significantly increased the dry matter yield of corn plant following wheat from 3.31 t ha^?1 (at control) to 7.89 t ha^?1 (at the TW treatment of 40 t ha^?1). The ¹⁵N values derived from the ¹⁵N fertiliser decreased with increasing TW application. The average values of N derived from N fertiliser (Ndff) varied from 2.14 to 3.09% at the rates of 20 and 80 kg N ha^?1, respectively. However, N derived from TW (Ndftw) significantly increased from 16.93 to 24.59% (at 20 kg N ha^?1), and it also increased from 23.06 to 28.15% (at 80 kg N ha^?1) with increasing TW applications from 20 to 40 t ha^?1, respectively.     

Development and application of 15N-tracer substances for measuring the whole-body protein turnover rates in the human,especially in neonates: a review

Our research group of the Children's Hospital of the University of Rostock (Rostock group) has long-time experience in ¹⁵N-labelling and in using yeast protein and its hydrolysates for tracer kinetic studies to evaluate parameters of the whole-body protein metabolism in premature infants. The particular advantage of applying an economically convenient, highly ¹⁵N-enriched, and completely labelled yeast protein for evaluating protein turnover rates is the fact that the ¹⁵N dose is spread among all proteinogenic amino acids. The absorption has been improved by hydrolysing [¹⁵N]yeast protein with thermitase into a mixture of amino acids, dipeptides and tripeptides so that faecal analysis becomes unnecessary when determining turnover rates. The review shows that, in contrast to the application of single ¹⁵N-labelled amino acids with resulting overestimation of protein turnover rates, the ¹⁵N-labelled yeast protein thermitase hydrolysate represents the amino acid metabolism more closely without causing amino acid imbalances. The ¹⁵N-labelled yeast protein thermitase hydrolysate leads to the estimation of reliable protein turnover rates, particularly in premature infants.     

Computerprogramm zur interaktiven Auswertung von 15N-tracerkinetischen Stoffwechselexperimenten

The theoretical concept of the computer program is based on the compartment theory for the 3-pool model using single pulse administration of the tracer. The code estimates the model parameters by means of the non-linear method of least squares fit under steady state conditions. Furthermore the parameters of the protein metabolism are calculated. The program works interactively and allows reading and modifying the experimental ¹⁵N tracer data via terminal and controlling the program.     

Measuring of the Integral Airborne Nitrogen-Input into a Soil-Plant System by the 15N-Isotope Dilution Method

Abstract

Airborne nitrogen-inputs so far have only been measured in single fractions (deposition on plant surfaces or on soil and direct absorption of nitrogen containing gases by plants) by intensive exposition experiments in gas chambers, measurement of wet and dry N-deposition in the field and very expensive micrometereological field measurements. It is very difficult to estimate any overall N-input with practical relevance from these single N-component measurements. In this introduced field experiment an isolated measuring system is labelled with a ¹⁵N-tracer since it is not possible to label the nitrogen compounds of the atmosphere (e.g. NH₃, NO_x) which are to be absorbed. Through the dilution of this ¹⁵N-tracer by nitrogen derived from the atmosphere the total input of airborne nitrogen is determined. As soil resembling substrate sand was used and summer wheat was planted. With the regular and automated irrigation of nitrogen-free nutrient solution and the collection of precipitation surpluses this system measured the total input of airborne nitrogen for one vegetation period.

The first application of the described system gave an airborne nitrogen input into the soil-plant system during a vegetation period of 10 weeks (April-June) of about 32 kg ha^?1.     

Isotope variations of hydrogen,carbon and nitrogen in florae from the Schirmacher Oasis,East Antarctica

Comparative biochemical and isotope-chemical investigations of cosmopolitical plants open up ways of obtaining parameters from different parts of the Earth which are characterised by variations in the habitat due to different environments. As an Antarctic oasis, the Schirmacher Oasis disposes of adequate favourable ecological conditions for the growth of lower plants.

In the present paper, results of isotope studies of lichens, mosses and algae of the Schirmacher Oasis are given and peculiarities of the habitats which influence the isotope contents of the plants are discussed.     

The metabolic effect of resistant starch and yoghurt on the renal and faecal nitrogen and ammonia excretion in humans as measured by lactose-[15N2]ureide

Resistant starch (RS) and Lactobacillus acidophilus yoghurt (LC1) were supplemented simultaneously in healthy adults to evaluate the effect on the urinary and faecal nitrogen and ammonia excretion by means of lactose-[¹⁵N₂]ureide (¹⁵N-LU) degradation. Nineteen subjects received a regular daily diet either without or with supplementation of an RS-LC1-mixture composed of fibre of potatoes (RS type 1), wrinkle pea starch (RS type 2), and LC1 over a 20-day period in randomised order. Thereafter, ¹⁵N-LU was administered together with breakfast. Urine and faeces were collected over a period of 48 and 72 h, respectively. The ¹⁵N abundances were measured by isotope ratio mass spectrometry. The intake of the pre- and probiotic mixture composed of RS of type 1, type 2 and of LC1 significantly lowered the colonic generation and the renal excretion of toxic ¹⁵NH₃ and functioned as an ammonia shift from urinary to faecal ¹⁵N excretion when using ¹⁵N-LU as a xenobiotic marker.     

Identification of the nitrate contamination sources of the Brusselian sands groundwater body (Belgium) using a dual-isotope approach

Isotopic fingerprinting is an advanced technique allowing the classification of the nitrate source pollution of groundwater, but needs further development and validation. In this study, we performed measurements of natural stable isotopic composition of nitrate (¹⁵N and ¹⁸O) in the groundwater body of the Brussels sands (Belgium) and studied the spatial and temporal dynamics of the isotope signature of this aquifer. Potential nitrogen sources sampled in the region had isotopic signatures that fell within the corresponding typical ranges found in the literature. For a few monitoring stations, the isotopic data strongly suggest that the sources of nitrate are from mineral fertiliser origin, as used in agriculture and golf courses. Other stations suggest that manure leaching from unprotected stockpiles in farms, domestic gardening practices, septic tanks and probably cemeteries contribute to the nitrate pollution of this groundwater body. For most monitoring stations, nitrate originates from a mixing of several nitrogen sources. The isotopic signature of the groundwater body was poorly structured in space, but exhibited a clear temporal structure. This temporal structure could be explained by groundwater recharge dynamics and cycling process of nitrogen in the soil-nitrogen pool.     

Dietary fibre-rich resistant starches promote ammonia detoxification in the human colon as measured by lactose-[15N2]ureide

Three resistant starches (RSs), namely fibre of potatoes (FP), wrinkle pea starch (WPS), and high amylose maize starch (HAMS) with different dietary fibre contents, were supplemented in adults to evaluate their effects on urinary nitrogen and ammonia excretion as well as on faecal nitrogen excretion by means of lactose-[¹⁵N₂]ureide (¹⁵N-LU) degradation. Twenty subjects received a regular diet either without or with the supplementation of FP, WPS, and HAMS in a randomized order. After administration of ¹⁵N-LU, urine and faeces were collected over 48 and 72 h, respectively, whereas blood was collected after 6 h. The ¹⁵N-abundances were measured by isotope ratio mass spectrometry. In comparison to the dry run, supplementation with RS significantly lowered renal ¹⁵N-excretion (dry run: 43.2?%, FP: 34.6?%, WPS: 37.9?%, HAMS: 36.4?%) as well as the corresponding ¹⁵NH₃-excretion (dry run: 0.08?%, FP: 0.06?%, HAMS: 0.05?%), clearly indicating a reduced colonic nitrogen generation at high dietary fibre intake.     

Estimation of protein synthesis rates using the flooding method and [15N] lysine

After injection of a single dose of double labelled lysine (1 MBq L-[u-¹⁴C]lysine and 150 μmol L-[α¹⁵N]lysine (95 atom% ¹⁵N)) to growing rats the fractional protein synthesis rates (FSR) of some organs were estimated by the “large dose-” or “flooding method”. Data obtained with both substances were compared and the following conclusions can be drawn:

—In principle lysine is suited as a flooding substance.

—In flooding experiments [¹⁴C]lysine and [¹⁵N]lysine gave identical FSR-values for the investigated organs.

—By application of [¹⁵N]lysine instead of the ¹⁴C labelled amino acid as flooding substance this method is suited for larger animals (pigs, sheep, ruminants) too, because of the absence of any radioactive burden.

Nach einmaliger Verabfolgung von doppelt markiertem Lysin (1 MBq L-[u-¹⁴C]Lysin und 150 μmol L-[α¹⁵N]Lysin (95 Atom% ¹⁵N)) an wachsende Ratten wurden nach der “Large dose-” oder “Flooding-Methode” die fraktionellen Proteinsyntheseraten (FSR) einiger Organe bestimmt und die für beide Substanzen erhaltenen Werte verglichen. Es ergaben sich folgende Schlußfolgergungen:

—Lysin ist grundsätzlich als Flooding-Substanz geeignet.

—[¹⁴C]Lysin und [¹⁵N]Lysin ergeben im Floodingversuch am gleichen Tier für die FSR der Organe identische Werte.

—Durch den Fortfall der radioaktiven Belastung bei Einsatz von [¹⁵N]Lysin anstelle von [¹⁴C]Lysin als Flooding-Substanz ist die Methode auch bei Croßtieren anwendbar.     

Stable isotopes of H,C and N in mice bone collagen as a reflection of isotopically controlled food and water intake*

²H/¹H ratios in animal biomass reflect isotopic input from food and water. A 10-week controlled laboratory study raised 48 mice divided in two generations (8 mothers Mus musculus and their offspring). The mice were divided into four groups based on the combination of ²H, ¹³C, ¹⁵N-enriched and non-enriched food and water. Glycine, the most common amino acid in bone collagen, carried the ²H, ¹³C, ¹⁵N-isotopic spike in food. ANOVA data analysis indicated that hydrogen in food accounted for ～81?% of the hydrogen isotope inventory in collagen whereas drinking water hydrogen contributed ～17?%. Air humidity contributed an unspecified amount. Additionally, we monitored ¹³C and ¹⁵N-enrichment in bone collagen and found strong linear correlations with the ²H-enrichment. The experiments with food and water indicate two biosynthetic pathways, namely (i) de novo creation of non-essential amino acids using hydrogen from water, and (ii) the integration of essential and non-essential amino acids from food. The lower rate of isotope uptake in mothers’ collagen relative to their offspring indicates incomplete bone collagen turnover after ten weeks. The variance of hydrogen stable isotope ratios within the same cohort may limit its usefulness as a single sample proxy for archaeological or palaeoenvironmental research.     

Evaluation of the oral 13C-bicarbonate tracer technique for the estimation of CO2 production and energy expenditure in dogs during rest and physical activity

For feeding of working dogs during their daily life, illness, routine jobs or sporting activities, an accurate determination of their nutritional requirements is essential to ensure their optimal health and performance. To predict the appropriate guidelines about how to feed dogs, it appears essential to determine the energy expenditure (EE) in a reliable and feasible way. In the present experiment, the non-invasive oral ¹³C-bicarbonate tracer technique (o¹³CT), i.e. collection of breath samples after oral administration of NaH¹³CO₃, was used for the estimation of CO₂ production and EE in dogs. Measurements were conducted during two days of rest, and during three days with 3 h of exercise per day. Average EE was 483 and 876 kJ kg^?0.75 d^?1 during rest and exercise, respectively. The o¹³CT seems appropriate to use as a minimal restrictive and non-invasive method to obtain reliable estimates of EE in dogs at different activity levels under near natural conditions.     

15N NMR of glycine metabolism in liver,muscle and kidney of rats using a new pulsed overhauser technique (POE)

Following intravenous injections of [¹⁵N]glycine (97 at.% ¹⁵N, 400 mg in 1.5 ml isotonic saline) to Lewis rats several organs were excised. The time courses of the ¹⁵N NMR spectral (40.55 MHz) were measured at 37°C after cold (4°C) or warm (37°C) storage. The application of the Overhauser POE technique yielded signal enhancement by a factor of -2.7 and -1.3 in sceletal and heart muscle, respectively, at 37°C, but no enhancement in liver and kidney.

In the time course of liver measurements at 37°C, intermediate metabolic products of glycine were observed, such as serine, glutamine, alanine, and tryptophane.     

Nitrogen isotopic signature of vegetables from the Slovenian market and its suitability as an indicator of organic production

The aim of this study was to obtain preliminary data on δ¹⁵N values in organically and conventionally grown vegetables available on the Slovenian market to determine whether there are any characteristic differences in δ¹⁵N values, which could be used as a marker to differentiate between organically and conventionally grown vegetables, namely vegetables grown with synthetic or organic nitrogen fertilisers. Therefore, 14 different varieties of organically and conventionally grown vegetables were analysed. The results obtained show clear differences in mean δ¹⁵N values between organically and conventionally grown vegetables (up to 6.3\permil). However, due to overlapping results, it was not possible to differentiate between organically and conventionally grown counterparts in eight vegetable varieties, despite the differences in mean δ¹⁵N values. The results obtained indicate that the method has several limitations. Hence, δ¹⁵N could be a reliable indicator for organic production, but nevertheless, the conclusion has to be validated by background information.     

Effect of alcohol consumption on whole-body protein turnover in healthy adults

The aim of the study was to investigate the whole-body protein turnover, either before or after continuous, moderate ethanol-induced oxidative stress by red wine consumption over a relatively short period in healthy volunteers. Ten healthy adults received an individual regular diet over 20 days. After 10 days, the subjects consumed 0.4 ml ethanol kg^?1 day^?1 as red wine together with dinner over a 10-day period. After 8 and 18 days, respectively, a ¹⁵N-labelled yeast protein was administered in a dosage of 4.2 mg kg^?1 body weight. Urine and faeces were collected over 48 h, respectively. The ¹⁵N-enrichment was measured by isotope ratio mass spectrometry, whereas the protein flux rates were calculated by a three-compartment model. The whole-body protein turnover without/with red wine consumption amounted to 3.73±0.6 and 3.49±0.6 g kg^?1 day^?1 (not significant), respectively. Moderate alcohol consumption does not induce significant short-term changes in the whole-body protein turnover of healthy adults.     

Impact of diazotrophy on N stable isotope signatures of nitrate and particulate organic nitrogen: case studies in the north-eastern tropical Atlantic Ocean

During two independent cruises in the north-eastern tropical Atlantic Ocean, we applied two different approaches to investigate the impact of diazotrophy on nitrogen stable isotope signatures in nitrate and particulate organic nitrogen (PON) of the food-web constituents. The first approach, used during the Poseidon cruise 348 in the Mauritanian upwelling, investigated the long-term influence of diazotrophy on the natural abundance of δ¹⁵N-NO^? ₃ and PON. The second approach, adopted during the Cape Verde field cruise, applied stable isotope tracer addition experiments. These served to determine the instantaneous transfer of diazotrophic N to the higher trophic level. Both approaches showed that N₂ fixation was compatible with the pattern and the magnitude of the isotopic depletion of dissolved NO^? ₃ during the Mauritanian upwelling cruise, as well as PON in zooplankton and phytoplankton during the Cape Verde cruises. An N-budget using ¹⁵N incorporation rates and diazotrophic N₂ fixation rates showed that 6 % of the daily N₂ fixation was potentially taken up by the mesozooplankton community. Direct grazing accounted for 56 % of gross mesozooplanktonic N incorporation, while 46 % occurred due to channelling through the microbial loop.     

Aufnahme von 15NO2 und Einbau des 15NO2-Stickstoffs in verschiedene Stickstofffraktionen bei Sonnenblumen (Uptake of 15NO2 and Metabolic Transfer of the 15NO2 Nitrogen to Various Nitrogen Fractions of Sunflowers)

Abstract

Sunflowers were exposed to ¹⁵NO₂ at a range between 4,9 and 42,0 ppb. The ¹⁵NO₂ uptake was quantified and the fate of the ¹⁵N in different parts and pools of nitrogen was investigated. The very high δ¹⁵N-values of the free amino acid pool can't give an answer to the question whether the NO₂-nitrogen is incorporated by a different pathway, compared to the nitrogen which derives from root uptake. Or does a compartmentation of the nitrate pool in the plant cell cause a dilution of the ¹⁵N enrichment in the mineral nitrogen pool during sample preparation?     

15N natural abundance of non-fixing woody species in the Brazilian dry forest (caatinga)

Foliar δ¹⁵N values are useful to calculate N₂ fixation and N losses from ecosystems. However, a definite pattern among vegetation types is not recognised and few data are available for semi-arid areas. We sampled four sites in the Brazilian caatinga, along a water availability gradient. Sites with lower annual rainfall (700 mm) but more uniform distribution (six months) had δ¹⁵N values of 9.4 and 10.1 ‰, among the highest already reported, and significantly greater than those (6.5 and 6.3 ‰) of sites with higher rainfall (800 mm) but less uniform distribution (three months). There were no significant differences at each site among species or between non-fixing legume and non-legume species, in spite of the higher N content of the first group. Therefore, they constitute ideal reference plants in estimations of legume N₂ fixation. The higher values could result from higher losses of ¹⁵N depleted gases or lower losses of enriched ¹⁵N material.