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 (15)N-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 (15)N-enriched, and completely labelled yeast protein for evaluating protein turnover rates is the fact that the (15)N dose is spread among all proteinogenic amino acids. The absorption has been improved by hydrolysing [(15)N]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 (15)N-labelled amino acids with resulting overestimation of protein turnover rates, the (15)N-labelled yeast protein thermitase hydrolysate represents the amino acid metabolism more closely without causing amino acid imbalances. The (15)N-labelled yeast protein thermitase hydrolysate leads to the estimation of reliable protein turnover rates, particularly in premature infants.     

Zum Stand der Stoffwechselforschung mit 15N- und 13C-markierten Tracersubstanzen in der Kinderheilkunde (Protein Turnover Research Using 15N and 13C Labelled Substrates in Pediatrics)

Abstract

Measurements in protein turnover and in metabolism of amino acids and their degradation products by means of stable isotope labelled substrates have been increasingly applied in clinical research over the last years. In spite of numerous studies dealing with this topic, quite a few important insufficiently clarified methodical aspects remain. This refers, for instance, to the choice of suitable tracer substances, the difficulties in the determination of the excretion plateau and the validation of the oxidation rates as measured with individual-labelled amino acids with regard to the whole body protein synthesis. Such problems may become of decisive importance in special subjects, such as preterm infants and critically-ill patients.

Investigations into these issues conducted by our group have revealed that the protein turnover in the very small preterm infant is by no means as intensive as previously claimed. The utilisation of urea nitrogen for the whole body protein synthesis of the infant may assume substantial proportions under the conditions of marginal protein intake and of catchup-growth. Studies conducted by means of ¹⁵N-labelled bifidobacteria have pointed at the intensive substrate exchange existing between microflora and host.

Pediatric research has to be non-invasive. Consequently, methods based on arterio-venous differences in tracer concentrations and on muscle biopsies do not have very high priority in pediatric research. A search for references published in the last five years has shown, that ¹⁵N-glycine is still the most frequently used tracer substance. There is a tendency towards a further increase of cell culture experiments run with stable isotope labelled amino acids.

Clinical research groups increasingly turn their attention to stable isotopes and mass spectrometry. This impressively demonstrates the continuing importance of tracerkinetic methods in all branches of medicine.     

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.     

Influence of Food Restriction and Compensatory Growth on Protein Synthesis and Breakdown in Growing Rats

The experiment was carried out with 24 male albino rats (5 weeks old, live weight 79 ± 5.1 g) divided into 2 groups. Gr. 1 (control group) was fed the commercial breeding food Ssniff with 22.4% crude protein ad lib., Gr. 2 (test group) for the first fortnight a mixture of the breeding food/cellulose 60/40 (restriction period) and subsequently for the second fortnight (compensatory period) also the breeding food ad lib. During both of these periods an 8 day N balance trial and a 4 day ¹⁵N tracer trial were included for estimation of growth rate and protein synthesis rate. Protein synthesis was ascertained by the endproduct method (oral application, single dose, mixture of 12 ¹⁵N-labelled amino acids); protein breakdown was calculated as the difference of protein synthesis rate and growth rate. The estimated rates were converted into fractional ones by referring to the body N content of corresponding animals. From the results of this experiment is concluded: The smaller N balance during moderate nutritional restriction can be attributed to a decrease of protein synthesis. The compensatory growth of the animals during the realimentation period is caused by an increase of protein synthesis and the enhanced protein conversion. Protein degradation is nearly unaffected.     

Diskriminierung der Stickstoff-Isotope im Leber-Metabolismus nach Verzehr von Unterschiedlichen Nahrungsproteinen

Nitrogen isotope ratio disproportionation (N-IRD) is the result of isotope effects of different magnitude in branched pathways of nitrogen metabolism of the liver. N-IRD is measured as the difference of natural ¹⁵N abundance of urea and plasma protein and is noted in ‰ (per mill) as the difference of their δ values.

It was derived that N-IRD would become maximum if the amino acid pattern consumed by the respective pathways urea synthesis and protein synthesis of the liver is identical. This is expected if the metabolic available amino acid pattern exactly meets requirement.

When feeding rats methionine(met)-supplemented casein N-IRD increased from 6‰ at zero protein intake to 8‰ at the requirement intake of 5 g/kg BM?)/day. With further increasing protein intake N-IRD decreased to values below 6‰. It was concluded that increasing N-IRD with increasing intake of met-supplemented casein is caused by complementation of the endogenous amino acid pattern by exogenous dietary amino acids with a complementation optimum at protein intake of 5 g/kg BM/day where N-IRD was maximum.

When feeding rats soy protein N-IRD (= 6.2‰) did not increase, but declined slightly above requirement intake of 10 g/kg BM/day to N-IRD = 5.7‰ at 30 g/kg BM/day. This indicates that the amino acid pattern of the metabolic pool was not complemented by intake of soy protein.

These findings strongly suggest that the (endogenous) amino acid pattern of the metabolic pool, as present at zero protein intake, is characterized by an imbalance (“endogenous inbalance”) which may be complemented dependent on the intake of appropriate protein.     

Enzymatic synthesis of some 15N-labelled l-amino acids

Our group has developed a stereospecific enzymatic method, which is very efficient for the in vitro synthesis of l-[¹⁵N]serine, l-[¹⁵N]methionine and l-[¹⁵N]glutamic acid. These amino acids were prepared from the corresponding α -ketoacids in the suitable enzymatic systems. The bacterial NAD-dependent amino acid dehydrogenases alanin dehydrogenase, leucin dehydrogenase and glutamate dehydrogenase were used as catalysts. Glucose dehydrogenase was used for the regeneration of NADH and ¹⁵NH₄Cl as isotopically labelled material at 99 at.% ¹⁵N. All reactions are inexpensive and easy to perform on a synthetically useful scale (1-10g) giving high yields of l-amino acids. The ¹⁵N isotope content was determined by mass spectrometry.     

Präparative Darstellung von hochangereichertem 15N-markiertem Hefeprotein

A method of biological preparation of highly-enriched yeast protein labelled ¹⁵N is described. The special relevance of such completely labelled proteins results from their importance for tracer-kinetic investigations of metabolism.     

Relationships between Nitrogen Translocation and Accumulation in Growing Plant Parts of Grain Legumes

Abstract

Grain legumes absorbed mineral ¹⁵N at every stage of development and transported it directedly (just as symbiotically fixed ¹⁵N₂) into the plant parts growing at the respective stage. The ¹⁵N accumulation in the grains after long-lasting ¹⁵N supply can be ascribed, for the major part, to a secondary ¹⁵N translocation after a temporary incorporation into older plant parts (leaves, stem). Inhibition experiments with antibiotics revealed no direct relation between the accumulation of amino acid ¹⁵N in growing pods and seeds and the protein synthesis in this target organs. It may include, however, processes of (active ?) uptake and transport with a possible contribution of carrier systems specific for distinct amino acids.     

The Influence of Feeding Frequency on Nitrogen Turnover and Whole Body Protein Synthesis in Adult Rats (Estimation using [14C]- and [15N]- Labelled Leucine)

Abstract

Male Wistar rats (17 wks. old, body weight ～400 g), fitted with an intra gastric cannula and with a catheter in the vena jugularis were divided into 3 groups and given a marginal ration of the feeding solution Nutrison Standard (1g protein and 350 kJ ME per day). Group 1 had ad lib. access to the drinking bottle, the groups 2 and 3 were pair fed by gastric infusion, splitted up into 2 greater meals for group 2 respectively into 6 smaller meals for group 3. After adaptation all animals get an i.p. injection of doubly labelled tracer solution (200μl) containing 2.5mg L-[¹⁵N]leucine (72 atom% ¹⁵N) combined with either [1-¹⁴C]- or [U-¹⁴C] leucine (37 kBq).

The course of ¹⁴CO₂ expiration was estimated by breath test over 4h in intervals of 15 min and the course of urinary ¹⁵N excretion over 24h in intervals of 45 resp. 90 min. An infusion of saline (0.9% 5ml/h) into the vena jugularis was used to provoke sustained urine production of the animals during the experiment.

From the parameters of the excretion curves of breath ¹⁴CO₂ resp. urine ¹⁵N (cumulative end value) and from the N balance the portions of leucine-C and leucine-N used for protein synthesis, transamination decarboxylation and total oxidation as well as the kinetic parameters for whole body protein metabolism were computed.

The following conclusions were drawn:

6 x feeding regime produces a small but measurable amino acid economy effect in comparison to 2 x feeding regime.

Protein gain for 2 x feeding group was significant smaller than for 6 x feeding group, though protein synthesis rate was higher, but was overcompensated by a greater increase of protein breakdown rate for the 2 x feeding group. Energy storage in form of fat and glycogen built from decarboxylation was unaffected by feeding frequency. The amount of leucine oxidized for heat production was 4% higher for the 6 x feeding group. Transamination rate for leucine was estimated to 8–15%. Absolute values for protein flux, protein synthesis and protein breakdown may be overestimated or underestimated because the metabolism of [¹⁵N] leucine does not exactly agree with that of total N; but the proportions of them and therefore also the conclusions will be true. Better results for absolute values will be obtained using a mixture of ¹⁵N labelled AA, ¹⁵N labelled protein or hydrolysate of ¹⁵N labelled protein (yeast) as the tracer source.     

Maternal obesity does not influence human milk protein 15N natural isotope abundance

ABSTRACT

Obesity increases protein metabolism with a potential effect on nitrogen isotope fractionation. The aim of this study was to test the influence of obesity on human milk extracted protein ¹⁵N natural isotope abundance (NIA) at one month post-partum and to compare human milk extracted protein ¹⁵N NIA and bulk infant hair ¹⁵N NIA. This cross-sectional observational study involved 16 obese mothers (body mass index (BMI)?≥?30?kg?m^?2 before pregnancy) matched with 16 normal-weight mothers (18.5?kg?m^?2?≤?BMI?<?25?kg?m^?2) for age and pregnancy characteristics. Human milk extracted protein and bulk infant hair ¹⁵N NIA were determined by isotope ratio monitoring by mass spectrometry interfaced to an elemental analyser (IRM-EA/MS). No significant difference was found in human milk protein ¹⁵N NIA values between obese and normal-weight mothers (8.93?±?0.48?‰ vs. 8.95?±?0.27?‰). However, human milk protein ¹⁵N NIA was significantly lower than bulk infant hair ¹⁵N NIA: 8.94?±?0.38?‰ vs. 9.66?±?0.69?‰, respectively. On the basis of these results, it is concluded that human milk protein ¹⁵N NIA measured at one month post-partum is not influenced by maternal obesity. These findings suggest that ¹⁵N NIA may be exploited to study metabolism without considering maternal obesity as a confounder.     

Amino Acid 15N/14N Analysis at Natural Abundances: A New Tool for Soil Organic Matter Studies in Agricultural Systems

Abstract

The effects of landuse, fertilizer history and soil type on the quantity and isotopic quality of hydrolysable soil amino acids were examined in 3 grassland and 2 arable soils. Results showed, (i) that overall concentrations of individual amino acids were highest in the grassland soils, (ii) that ‰δ¹⁵N values of the individual amino acids differed considerably between the five soils, and (iii) that the combination of amino acid ‰δ¹⁵N values and concentrations could be used to distinguish between landuse, crop type and fertilizer history. This preliminary study indicates that the pathways of transformation of soil amino acid N are influenced by long term N inputs and that associated biological processes are reflected in differences in concentrations and ‰δ¹⁵N values of individual soil amino acids.     

Eignung verschiedener 15N-Verbindungen als Tracer für Körpermarkierung und für Untersuchungen des Protein- und Aminosäuremetabolismus

In einem Versuch mit wachsenden Ratten werden die Resorption, die Retention und die intermediäre Verteilung des ¹⁵N nach zehntägiger Verabfolgung von vier verschiedenen ¹⁵N-Tracern: DL-(¹⁵N)-Methionin, L-(¹⁵N)-Lysin, (¹⁵N)-Glycin und (¹⁵NH₄)₂SO₄ untersucht und die Übertragungsprozesse der markierten Aminogruppe (Transaminierung, Desaminierung und Aminierung sowie andere chemische Umwandlungen zwischen den Aminosäuren) bestimmt. Die Ergebnisse werden diskutiert und daraus Schluβfolgerungen für die Eignung der Substanzen zur Markierung von Organismen bzw. als Tracer für die Bestimmung von Parametern des Protein- und Aminosäurenstoffwechsels abgeleitet.

In an experiment with growing rats the resorption, the incorporation and the intermediate fate of ¹⁵N were estimated using four different ¹⁵N-tracers: DL·(¹⁵N)-methionine, L·(¹⁵N)-lysine, (¹⁵N)-glycine and (¹⁵N₄)₂SO₄. Transformation of the labelled amino group by transamination, by desamination and amination as well as by other chemical processes of amino acids will be checked. The results are discussed and conclusions are drawn for the use of ¹⁵N -substances as a labelling substance of organisms respectively as a tracer for estimating parameters of the whole body protein and amino acid metabolism.     

Whole Body Protein Metabolism Estimated by 15N Tracer Experiments and Body Composition of Mice Selected for Different Growth Parameters

Abstract

Whole body protein synthesis was investigated in growing male mice which were long-time selected for high carcass protein amount (DU-6P, protein line) or for high body weight (DU-6, growth line) and in the unselected randomly bred control (DU-Ks). Six mice/line were housed singly in metabolic cages for the estimation of N balance, whole body protein synthesis (end-product method, single dose of ¹⁵N-labelled amino-acid mixture), and N distribution in the body. Another six mice/line were used for the determination of the body composition. All mice had free access to a commercial stock diet (crude protein 268 g, gross energy 19 MJ/kg dry matter) and to water. Body weight of both selection lines was about twice that of control mice at the same age. Selection for high body weight resulted in higher body fat content. Scaled to the corresponding body protein pools, the protein synthesis rates of selected mice were significantly higher than in controls, but were not significantly different between both selection lines in contrast to the protein deposition rates. The higher protein accretion in the protein line in comparison to the growth line seems to be due to a combination of a lower protein breakdown and an increased protein synthesis rate.     

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.     

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.     

Long term changes in the distribution and δ15N values of individual soil amino acids in the absence of plant and fertiliser inputs

The long-term ‘biodegradation’ on soil amino acids was examined in the control plots of ‘42 parcelles’ experiment, established in 1928 at INRA, Versailles (France). None of the plots is cultivated, but is kept free of weeds, and mixed to a depth of 25 cm twice yearly. Topsoil (0–10 cm depth) samples collected in 1929, 1963 and 1997 were subjected to acid hydrolysis (6 N HCl) for comparison. The distribution and δ¹⁵N natural abundance of 20 individual amino acids in the soils were determined, using ion chromatography (IC) and gas chromatography–combustion–isotope ratio mass spectrometry (GC–C–IRMS). The total N and amino acid-N (AA-N), respectively, decreased by 54 % and 73 % in the period from 1929 to 1997. The average N loss was comparable for 1929–1963 (period 1) and 1963–1997 (period 2), but AA-N loss was three times faster in the former period. This significant reduction in total AA-N content was mirrored in the individual amino acids, which decreased by 74 %?±?1 % (ranging 58–89 %) between 1929 and 1997. The bulk δ¹⁵N values generally increased from 1929 to 1997, mainly associated with comparable or even higher increase of δ¹⁵N of the non-AA-N in the soil. The residence time (t _1/2, time in which half of N was lost from a specific soil pool) was ca. 65?±?5 years for the bulk soil, and comparable for periods 1 and 2. However, between periods 1 and 2 it decreased from 128 to 41 years in the non-AA pool, but increased from 59 to 92 years in the AA-N pool. Proline and amino acids that appear early in soil microbial metabolic pathways (e.g. glutamic acid, alanine, aspartic acid and valine) had relatively high δ¹⁵N values. Phenylalanine, threonine, glycine and leucine had relatively depleted δ¹⁵N values. The average δ¹⁵N value of the individual amino acids (IAAs) increased by 1δ unit from 1929 to 1997, associated with a similar rise from 1929 to 1963, and no change thereafter till 1997. However, the δ¹⁵N values of phenylalanine decreased by more than 7δ¹⁵N units between 1929 and 1997. The δ¹⁵N shift of IAAs from 1929 to 1963 and from 1929 to 1997 was not influenced by the relative amount of N remaining compared with the 1929 soil concentrations. The only exception was phenylalanine which showed decreasing δ¹⁵N associated with its decreasing concentration in the soil. We conclude therefore that in the absence of plant and fertiliser inputs, no change in the δ¹⁵N value of individual soil amino acids occurs, hence the original δ¹⁵N values are preserved and diagnostic information on past soil N (cycling) is retained. The exception was phenylalanine, its δ¹⁵N decreased with decreasing concentration from 1929 to 1997, hence it acted as a ‘potential’ marker for the land use changes (i.e. arable cropping to a fallow). The long term biological processing and reworking of residual amino acids resulted in a (partial) stabilisation in the soil, evidenced by reduced N loss and increased residence time of amino acid N during the period 1963–1997.     

Gel-Electrophoresis and Subsequent Optical Emission 15N Analysis to Identify 15N-Labelled Protein Fractions

Abstract

A combined procedure to detect of ¹⁵N/¹⁴N isotope ratios by emission spectrometric analysis after starch gel-electrophoresis was developed. ¹⁵N-labelled proteins of human serum were used to optimise this method. Electrophorised gel slices with protein fractions were directly digested for subsequent isotope analysis. This method is proposed for use in routine analysis for clinical application.     

Anthropogenic changes of nitrogen loads in a small river: external nutrient sources vs. internal turnover processes

Anthropogenic nutrient inputs increase the N-load in many aquatic systems, leading to eutrophication and potential changes of biological N-retention capacity. In this study, nitrate inputs in a small river were investigated along a gradient of anthropogenic influence. We aimed to determine changes in nitrate load and isotope signatures in the water column and to identify the anthropogenic influence on biological nitrogen assimilation and nitrification or denitrification in sediments. In seasonal sampling campaigns, we analysed dissolved inorganic nitrogen concentrations, and stable isotopes of nitrate. To differentiate rates of nitrate production and consumption in the pristine vs. agricultural river section, intact sediment cores were incubated with ¹⁵N-labelled nitrate. δ¹⁵N values of nitrate in the pristine river section were low, reflecting natural sources, but, as expected, increased with nitrate concentration in all seasons along the gradient. In general, nitrate retention and consumption were higher in the anthropogenically impacted than in the pristine river section, and nitrate consumption exceeded production. In addition to our measurements, modelled results also show that even in a small river, the anthropogenically enhanced consumption capacity is overwhelmed by surplus N-inputs, and nitrate consumption cannot increase in turn with external loads.     

Whole-body protein turnover in nephrotic subjects: A comparison between different degrees of proteinuria

In contrast to the increased hepatic albumin synthesis in subjects with proteinuria, little is known about the corresponding whole-body protein turnover rates (WPTR). The WPTR and the reutilisation rates (R) were investigated in 20 patients divided in three groups of different degrees of proteinuria (groups I–III: < 1, 1–3, > 3 g/m²/day, respectively). [¹⁵N]glycine was administered as a single oral pulse. Urine samples were taken over 2 days. After removing urinary proteins by trichloracetic acid, ¹⁵N-enrichment in the supernatant was measured by isotope ratio mass spectrometry. A three-compartment model was used to calculate WPTR and R, which showed a statistically significant difference between groups I and III (2.64 vs. 4.63 g/kg/day, and 70.4% vs. 80.8%, respectively, P < 0.01), whereas the net protein gain remained unchanged (0.13 vs. 0.22 g/kg/day). The higher the protein loss the higher the WPTR and the corresponding R. The severe protein loss provokes increased WPTR and R as well.     

Precise δ13C-determination in the Range of Natural Abundance on Amino Acids from Protein Hydrolysates by Gas Chromatography - Isotope Ratio Mass Spectrometry

Abstract

Routine on-line ¹³C-analysis by coupled gas chromatography - combustion – isotope ratio - mass spectrometry of individual amino acids out of mixtures in the natural abundance range demands their strictly standardized derivatization. For the N-acetyl-propylesters of amino acids from protein hydrolysates and blood serum a mean precision of 0.5% ° (1 SD) for derivatization, separation and measurement could be attained. From δ¹³C-values of derivatives δ¹³C-values of amino acids could be calculated by a carbon balance equation, implying an isotope effect on the C-1 of acetate of about 1.04 for nine amino acids. The global δ¹³C-value of casein calculated from the individual amino acids differed by less than 1.5%° from the directly determined value. The δ¹³C-values of amino acids from plant protein hydrolysates analyzed by the on-line method were in agreement with results obtained by classical procedures. The method permits the δ-value determination of up to nine amino acids from 200 μg of a mixture in less than one hour. By this means the detection of ¹³C-labeled amino acids in nmole amounts diluted by the 5000-fold amount of carrier becomes possible.