Transamination Governs Nitrogen Isotope Heterogeneity of Amino

Article pubs.acs.org/JAFC

Transamination Governs Nitrogen Isotope Heterogeneity of Amino Acids in Rats Alexander Braun,† Armin Vikari,‡ Wilhelm Windisch,‡ and Karl Auerswald*,† †

Lehrstuhl für Grünlandlehre, Department of Plant Science and ‡Fachgebiet für Tierernährung und Leistungsphysiologie, Technische Universität München, D-85350 Freising, Germany ABSTRACT: The nitrogen isotope composition (δ15N) of different amino acids carries different dietary information. We hypothesized that transamination and de novo synthesis create three groups that largely explain their dietary information. Rats were fed with 15N-labeled amino acids. The redistribution of the dietary 15N labels among the muscular amino acids was analyzed. Subsequently, the labeling was changed and the nitrogen isotope turnover was analyzed. The amino acids had a common nitrogen half-life of ∼20 d, but differed in δ15N. Nontransaminating and essential amino acids largely conserved the δ15N of the source and, hence, trace the origin in heterogeneous diets. Nonessential and nontransaminating amino acids showed a nitrogen isotope composition between their dietary composition and that of their de novo synthesis pool, likely indicating their fraction of de novo synthesis. The bulk of amino acids, which are transaminating, derived their N from a common N pool and hence their δ15N was similar. KEYWORDS: amino acid, 15N, transamination, isotopic scrambling

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INTRODUCTION

regulates nitrogen exchange beyond de novo synthesis and thus alters the δ15N even of (at least some) essential amino acids. Transamination by aminotransferase transfers the amino nitrogen between an amino acid and an α-keto acid; in turn, the α-keto acid becomes an amino acid and vice versa.6 Since most transamination reactions have equilibrium constants close to 1, the direction of a transamination reaction proceeds in large part as a function of the intracellular concentrations of the reactants.8 Transaminations can also be chained, providing a “nitrogen shuttle” even between amino acids of different functional groups (e.g., essential and nonessential).9,10 For example, L-serine:glyoxylate aminotransferase (transamination enzyme, EC 2.6.1.45) catalyzes the reversible amino transfer from serine to glycine and glycine:2-oxoglutarate aminotransferase (EC 2.6.1.4) catalyzes the reversible amino transfer from glycine to glutamate (Figure 1A). Glutamate, as the central amino acid in metabolism,6 is in turn linked to other transamination chains, like the branched-chain amino acid metabolism that links the essential amino acids leucine, isoleucine, and valine.12 Such chains provide continuous redistribution of amino nitrogen among transaminating amino acids beyond de novo synthesis.8 Hence, amino acids, which are subject to transamination and which we term transaminating amino acids, virtually share one common amino nitrogen pool.13 This should lead to a reduced heterogeneity in δ15N among transaminating amino acids when the δ15N of the diet is heterogeneous. However, since transamination implies an isotope effect, the δ15N of transaminating amino acids is not completely equal.14

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The nitrogen isotope composition (δ N) of organisms provides information on trophic systems.1,2 While the bulk δ15N of tissues indicates the trophic position of organisms,3,4 the δ15N of specific compounds has the potential to provide more detailed insights, because different compounds differ in biosynthetic pathways and thus may reveal isotope information from different (dietary) sources.5 This is particularly true for amino acids, as they provide the main nitrogen reservoir of bulk materials, but have individual biosynthetic pathways.6 Thus, alanine, aspartic acid, and glutamic acid (among others) reflect a similar trophic position as bulk tissues, whereas glycine and phenylalanine provide an accurate determination of the δ15N at the base of the food web,2,7 although reasons for this grouping, as well as the information that can be obtained from other amino acids, remain unknown. Understanding how the major biochemical mechanisms that govern nitrogen input and distribution, namely de novo synthesis and transamination, affect the δ15N of amino acids would shed light on these knowledge gaps. Additionally, the time window through which such isotope information in amino acids is retained in organisms and differences between amino acids in such time windows are worthy of investigation. For carbon isotope composition (δ13C), the amino acids’ ability for de novo synthesis largely explained the information that can be retrieved from their δ13C.2,7 Essential amino acids remain biochemically unmodified from diet to consumer and thus represent the δ13C of the amino acids in the diet. For example, leucine (essential) was not enriched in 13C between diet and consumer, whereas glutamic acid (nonessential) became enriched in 13C. Remarkably, both amino acids were enriched in 15N,7 indicating that the classification into essential/nonessential only applies for the carbon backbone but not for δ15N, because a biochemical process interfered that © 2014 American Chemical Society

Received: Revised: Accepted: Published: 8008

May 16, 2014 July 16, 2014 July 18, 2014 July 18, 2014 dx.doi.org/10.1021/jf502295f | J. Agric. Food Chem. 2014, 62, 8008−8013

Journal of Agricultural and Food Chemistry

Article

Figure 1. (A) Amino acid nitrogen pools and transfers in mammal metabolism. Amino acids (AAs) are given in the standard three-letter code (see Table 1); essential AAs are in boxes, and AAs analyzed in this study are underlined. A colored background, orange for essential and blue for nonessential AAs, means no transamination. The reactions are indicated in italics; they are succeeded by a number in parentheses indicating the reference (1, Berg et al.;6 2, Tomé and Bos;16 3, Hutson et al.;12 4, Matthews;15 5, Nakada;10 6, Voet and Voet11); TA = transamination (aminotransferation with αKA = α-keto acid) after AA1 + αKA2 ↔ αKA1 + AA2, in most cases dealing with Glu and α-ketoglutaric acid; the double arrows indicate reversibility exclusively for this reaction. In this case, “equilibration” with the Glu pool is faster than any other reaction of the AAs or the αKAs. Irreversible TAs, indicated by a one-pointed arrow, may occur, when the αKA is rapidly further converted (Tyr) or when the TA does not concern the AS/αKA itself but a derivative (Lys and Ser). D = degradation to Glu, S = synthesis from Glu (via different pathways), DH = Glu dehydrogenase. (B) The atom fraction x(15N) of amino acids in diet and in muscle before diet switch. Amino acids are indicated by their one-letter code (see Table 1). The 95% confidence intervals had a similar size as the markers. Note the different ranges of x and y axes. (C) Reaction progress of the atom fraction x(15N) for the amino acids phenylalanine (Phe), serine (Ser), and proline (Pro) after the diet switch. Each of these amino acids belongs to one of the three hypothesized groups (panel A). Since the y-axis is logarithmic, similar linear slopes indicate similar turnover rates.

hence, amino acids should in the present context be divided into three groups: (i) transaminating amino acids should eventually reach a rather homogeneous δ15N irrespective of their isotope composition in the diet, because their amino nitrogen is exchanged with that of other transaminating amino acids (a common nitrogen pool); (ii) nontransaminating and essential amino acids should retain their δ15N from diet, because nitrogen is not exchanged; and (iii) nontransaminating and nonessential amino acids should have a δ15N that ranges between that of their dietary δ15N and that of the δ15N of the nitrogen donor pool that supplies de novo synthesis. The exchange rate by transamination could determine the nitrogen isotope turnover and thus the time window through which isotope information can be perceived. If this were the case, the nitrogen isotope turnover must differ between amino acids, in particular between transaminating and nontransaminating amino acids. The effect of transamination on nitrogen isotope turnover could, however, be superimposed by the continuous release of amino acids from protein turnover, in particular by the degradation of proteins by proteolysis. In that

Some amino acids differ in their ability to be transaminated. In mammals, tyrosine can be degraded but not synthesized by transamination, because the equivalent α-keto acid that would accept nitrogen and turn into tyrosine is unavailable.15 Hence, tyrosine can only be a nitrogen donor to the common nitrogen pool. The amino acids lysine,16 threonine,17 proline,18 and phenylalanine (except for the dysfunction phenylkentonuria6) do not transaminate at all. Due to their disability of bidirectional transamination, these amino acids are termed “non-transaminating” in this study. Since many diet reconstruction studies based on isotopes focus on feeding generalists (e.g., refs 2, 13) and feeding generalists likely have an isotopically heterogeneous diet, it should be investigated how the δ 15 N of isotopically heterogeneous amino acids changes from diet to consumer, after nitrogen redistribution by de novo synthesis and transamination. We hypothesize that the joint consideration of transamination and de novo synthesis can largely explain the variation in δ15N among amino acids in consumers. Since transamination is the final step in de novo synthesis, its effect on δ15N of amino acids should be of a major priority, and 8009

dx.doi.org/10.1021/jf502295f | J. Agric. Food Chem. 2014, 62, 8008−8013


Article

acids were supplied by Chemotrade Chemiehandelsgesellschaft mbH, Leipzig, Germany, except for threonine, which was supplied by Campro Scientific, Veenendaal, The Netherlands. However, in the mixture, aspartic acid and serine were substituted isonitrogenously by glycine, cysteine was substituted by methionine, and tyrosine was substituted by phenylalanine. Proline was not added as a 15N-enriched amino acid. These modifications led to a large variation in labeling of the individual amino acids in the diet, with the atom fractions of 15N ranging from 0.004 to 0.033 (Table 1). This pronounced labeling was chosen because any fractionation by the metabolism will be much smaller and can thus be neglected. For example, biosynthesis of glutamic acid by N transfer from aspartic acid to α-keto glutaric acid is accompanied by N isotope fractionation,14 but this will lower the atomic fraction only by ∼0.004 and thus does not interfere with the labeling, which caused an atom fraction in the labeled diet of 0.022. At day 50, the six-week “chase period” started and supplementation of the 15N-enriched amino acids was stopped in order to induce an exponential reaction progress in nitrogen isotope composition in the animals. To achieve reaction progress in less time, the protein content was increased to 2.4 g d−1 (while starch was accordingly decreased to 2.1 g d−1 20). All procedures and protocols were approved by the Animal Care and Use Committee of Technische Universität München. Sampling. Rats were sacrificed by decapitation under anesthesia (diethyl ether) in groups of four rats at day 39 of the equilibration period and on days 2, 7, 13, 20, 34, and 41 during the chase period to investigate the reaction progress of the isotope composition following the diet switch (Figure 1C). The animals were dissected to provide muscle tissue (musculus femoris and musculus quadriceps). The muscles were combined, homogenized with an Ultra-Turrax (IKALabortechnik Jahnke & Kunkel GmbH & Co KG, Staufen i. Br., Germany), rinsed with a solution of Hexane−2-propanol (3:2) to eliminate lipids, and filtrated. The fat-free muscle material was dried for 12 h at 70 °C and stored at −80 °C. The diet was collected daily, starting 5 days before the chase period to day 7 of the chase period and additionally on days 10, 13, 17, 22, 27, 34, and 41 of the chase period. For more details on the keeping and sampling, see the work of Braun et al.20 Isotope Analyses of Amino Acids. Isotope analysis was carried out at the Leibniz-Institut für Nutztierbiologie, Dummerstorf, Germany, following the method of Metges and Petzke.21 In brief, samples of 2.5−3.5 mg of muscle lyophilizate were double-washed with 10% trichloroacetic acid, acetone, and ether. Washed samples were hydrolyzed for 24 h with 2 mL of 6 N HCl at 110 °C and derivatized to their N-pivaloyl-isopropyl esters. To this end, 2 mL of CH2Cl2 was added to 3−8 μmol of amino acids, and the solution was dried and solved in 100 μL of pyridine. An aliquot of 100 μL of pivalochloride was added, the solution was acetylated (30 min, 60 °C) and cooled, and 2 mL of CH2Cl2 was added. The mixture was then passed through a 4 cm silica gel (silica gel 60, 200−400 mesh) column (4 mm i.d.) and the filtrate was dried under a stream of N2 before adding 100 μL of ethyl acetate. The samples were then analyzed for nitrogen isotope composition on a gas-chromatograph (HP 5890, Waldbronn, Deutschland), connected to a gas-isotope-ratio-mass spectrometer (Finnigan MAT, Bremen, Deutschland), via a GC combustion interface. The interface comprised a combustion furnace reactor filled with copper and nickel oxide and platinum (980 °C) and a reduction furnace filled with elemental copper (600 °C). Calculation of x(15N) of Dietary Amino Acids. In the following, we denote isotope composition as the atom fraction [x(15N)], because its use is preferred over the use of δ values for tracer and mixing calculations:22

case the nitrogen isotope turnover should be similar among all amino acids. In order to examine the effect of dietary heterogeneity in δ15N of amino acids on the δ15N of amino acids in the consumer involving the nitrogen isotope turnover of amino acids, we fed rats a diet with 15N-labeled amino acids and analyzed the δ15N of amino acids in muscle protein. Subsequently, we switched the δ15N of the amino acids in the diet to quantify their nitrogen isotope turnover rate. This was necessary to evaluate potential differences in half-lives among amino acids that could induce differences in δ15N due to different levels of labeling.

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MATERIALS AND METHODS

Animals and 15N Labeling. Eighteen juvenile female Sprague− Dawley rats (Rattus norvegicus, Berkenhout) were assigned to cages and maintained at 25 °C ambient temperature, 60% relative humidity, and a 12 h day−12 h night cycle for 13 weeks. All rats had ad libitum access to deionized water supplemented with 0.014% NaCl and received a diet of 8 g d−1 (dry mass), consisting of 2.2 g of sucrose, 0.6 g of coconut fat, 0.3 g of minerals, 0.2 g of cellulose, 0.1 g of sunflower oil, 0.02 g of vitamins, and 0.02 g of DL-methionine. Diets were supplemented with starch and casein depending on the nutritional requirements of the juvenile rats. During the first 7 weeks, termed the “isotope equilibration period”, the supplement was 3.2 g d−1 (days 1− 17), 3.5 g d−1 (days 18−34), and 3.7 g d−1 (days 35−49) starch and 1.3, 1.0, and 0.8 g d−1 casein, respectively. Digestibility of casein in young rats is close to 100%.19 A mixture of 12 L-amino acids that was similar to the amino acid profile of casein but highly enriched in 15N (atom fraction 0.95 or 0.99) was added, approximately at a ratio 1:100 to casein, which remained constant over time (Table 1). All amino

Table 1. Amino Acid Profile in Dietary Casein and in the LAmino Acid Labeling Mixture and Resulting Atom Fraction of 15N [x(15N)] in the Bulk Dieta content in bulk diet (g/100 g) amino acid alanine arginine asparagine aspartic acid cysteine glutamine glutamic acid glycine histidineb isoleucineb leucineb lysineb methionineb phenylalanineb proline serine threonineb tryptophanb tyrosine valineb

Ala Arg Asn Asp Cys Gln Glu Gly His Ile Leu Lys Met Phe Pro Ser Thr Trp Tyr Val

A R N D C Q E G H I L K M F P S T W Y V

from casein

from label mixture

x(15N)

5 2.5 4.1 2.2 0.8 7.7 9.3 2.2 1.9 5.7 9.1 7.2 3.8 3.9 9.8 7.4 5.7 0.7 4.2 7.3

0.02 0.01 0 0 0 0 0.18 0.07 0.03 0.05 0.08 0.04 0.05 0.05 0 0 0.05 0.01 0 0.06

0.008 0.007 0.004 0.004 0.004 0.004 0.022 0.033 0.016 0.012 0.012 0.009 0.020 0.016 0.004 0.004 0.011 0.017 0.004 0.011

x(15N) = n(15N)/(n(14 N) + n(15N))

a

The x(15N) of amino acids in the bulk diet is calculated from mass balance and a x(15N) of 0.004 for casein and 0.950 for the enriched amino acids except for Tyr and Try, for which x(15N) was 0.990. The name of the amino acids is followed by their three and one letter codes, respectively. bThis amino acid is essential for rats and most other mammals.

(1)

where n(14N) and n(15N) are the amounts of isotope 14N and 15N of element N, respectively. The x(15N) of the amino acids in the bulk diet was calculated according to the mass balance of an amino acid provided by casein and provided by the enriched amino acids of the labeling mixture as 8010



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x(15N) = (mcasx(15N)cas + mlabel x(15N)label )/(mcas + mlabel )

variation was small among replicates for all amino acids (∼2% of the respective mean), indicating equal labeling among rats. Turnover of Amino Acid 15N. After the diet switch the 15 x( N) of all amino acids exponentially approached the x(15N) of the unlabeled diet (Figure 2). The mean half-life of the

(2) where mcas and mlabel are the masses of the specific amino acid provided by casein and by the labeled amino acid mixture, respectively, while x(15N)cas and x(15N)label are the corresponding x(15N) values. Turnover Estimation and Statistical Evaluation. Turnover as quantified herein only applies for nitrogen but not for the carbon skeletons of the amino acids, which follow different pathways of recycling, degradation, and synthesis. A previous study that used the same animals had shown that the reaction progress of all organs including the muscle exhibited a common delay of 0.5 d following a diet switch, which likely was caused by the transit time in the digestive tract.20 Since neglecting a delay would lead to an underestimate in isotope turnover, the delay (0.5 d) was subtracted from the sampling times to provide a delay-corrected reaction progress. Since growth affects the apparent reaction progress20,23 and the muscle from which the amino acids were sampled had a growth rate of ∼0.7 g d−1,20 the effects of growth and turnover rate were disentangled from the delay-corrected reaction progress by fitting the function x(15 N)t = x(15 N)∞ − (x(15 N)∞ − x(15 N)0 )(1 − Δw/wt )(1 + r / g ) (3)

Figure 2. Half-lives of nitrogen in amino acids (quantified herein) and in muscle tissue (quantified in Braun et al.20), obtained from the same animals. The error bars indicate the 95% confidence intervals.

where x(15N)t is the atom fraction at time t, x(15N)∞ is the atom fraction of the asymptote at the end of the reaction progress, and x(15N)0 is the atom fraction at time zero (time of isotope switch plus delay). The parameter wt is the dry mass of an amino acid at time t and Δw is wt − w0, where the subscript 0 denotes time zero. The growth rate constant is denoted g and estimated for each amino acid specifically as 0.7/γ g d−1, where γ was the ratio of the mass of a specific amino acid to the mass of all amino acids in the muscle (0.7 g d−1 was the growth rate of the entire muscle; see above and in Braun et al.20). The ratio r/g gives the relative contribution of isotope turnover (r) and growth (g) to the apparent reaction progress. The half-life (t1/2) is then given by ln(2)/r. The parameter x(15N)0 was obtained by measuring the isotope composition at the end of the “equilibration period” (as described above), as this value characterizes the constant isotope composition during the delay and simultaneously the intercept of the following reaction progress model when delay time is subtracted. The standard errors of the fitting function for x(15N)0 and t1/2 were then converted to confidence intervals for p = 0.05 and a two-sided error. All data analyses were done in R 2.15.1.

nitrogen in amino acids was 20 d (13−27 d), and the nitrogen half-lives were not significantly different among amino acids, and they were not statistically different from the nitrogen halflife of the entire muscle. In particular, turnover was not different for essential/nonessential and transaminating/nontransaminating amino acids (Figure 1C).

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DISCUSSION We hypothesized that the joint consideration of transamination and de novo synthesis divides amino acids into three groups that largely explain the variation in x(15N) among amino acids in animals: (i) transaminating, (ii) nontransaminating and essential, and (iii) and nontransaminating and nonessential amino acids. Transaminating amino acids had a homogeneous x(15N) value in consumer tissues, despite their x(15N) value in the diet varying by an order of magnitude (Figure 1B). For example, serine was not provided by the labeled amino acid mixture but had the same x(15N) value in the muscle tissue as the labeled amino acids (Table 1). The x(15N) value of transaminating amino acids was similar to the x(15N) of the bulk diet (plus an expectable isotope enrichment24), and it was similar to the mean of x(15N) of transaminating amino acids in the diet. This indicated that complete redistribution of amino nitrogen among transaminating amino acids in consumer tissues can be expected, because transamination is the most common reaction involving free amino acids.17 However, the transaminations cause isotope fractionation25,26 that appears herein only as minor variation around the homogenized x(15N), because of the very high labeling. Future research should gain detailed knowledge on the isotope fractionations of the specific transamination enzymes. The harmonization of nitrogen isotope composition of transaminating amino acids seems to be consistent across trophic levels, environments, and diets, as similar nitrogen isotope compositions can be found for transaminating amino acids within herbivores, e.g., pig, and terrestrial and marine carnivores, e.g., lion and whale (see results of Hare et al.13). Thus, our results would be applicable to the many studies using

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RESULTS x(15N) of Amino Acids. At the end of the equilibration period, the x(15N) of amino acids varied by about a factor of 2 in muscle tissue, ranging from 0.0067 in tyrosine to 0.0158 in phenylalanine. The x(15N) of dietary amino acids did not correlate significantly with the x(15N) of muscular amino acids (p > 0.05, r2 = 0.29), but the following pattern was apparent (Figure 1B): (i) The transaminating amino acids alanine, glutamic acid, glycine, isoleucine, leucine, serine, and valine had a common x(15N) of 0.0108 (SD 0.0007), which was not statistically different from the mean x(15N) of the diet (0.0101 ± 0.0001). Hence, their x(15N) had changed substantially, including depletion and enrichment in 15N. The change ranged from −0.0211 in glycine to 0.0071 in serine. (ii) The x(15N) of nontransaminating and essential amino acids lysine, threonine, and phenylalanine was quite similar in diet and muscle; hence, they appeared on the 1:1 line (Figure 1 B). (iii) The nontransaminating and nonessential amino acids proline and tyrosine had a x(15N) between their initial (dietary) x(15N) and the x(15N) of their specific nitrogen donor pools, glutamine and phenylanaline, respectively. The coefficient of 8011



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N in mammals.27−30 This finding is, however, in contrast to x(15N) values in a zooplankter,7 indicating that the ability of transamination may differ between organisms that are more remote in their evolution and which do not share conserved biochemical pathways. This calls for the need to repeat such investigations in other animal groups that are in the focus of isotope animal ecology, e.g., birds or insects. Homogeneous x(15N) in consumer but varying x(15N) in diet implies variation in trophic shifts among transaminating amino acids, including enrichment and depletion in 15N, as found herein (Figure 1B) and in other studies.5,31 Amino acids become enriched in 15N when their dietary x(15N) is below the homogenized x(15N) and vice versa; the height of the trophic shift will be defined by the difference between isotope composition of an amino acid and the homogenized x(15N). Hence, the trophic shift of a transaminating amino acid cannot indicate the trophic position of the consumer. Nontransaminating and nonessential amino acids changed their x(15N) from diet to consumer to less extent. Considering their biochemical pathways, each amino acid of this group in consumer is either derived from its dietary amino acid pool (without nitrogen exchange by transamination) or synthesized de novo by the organism. Hence, the change in x(15N) should be a function of their dietary x(15N), the x(15N) of the donor pool supplying de novo synthesis, the fraction of de novo synthesized amino acids, and isotope fractionation during de novo synthesis:14 Principally, the x(15N) of these amino acids may retain their dietary x(15N) in the case of no de novo synthesis (e.g., when dietary supply exceeds the need); the x(15N) of these amino acids should be equal to the x(15N) of the precursor pool (±isotope fractionation) in the case of the fraction of de novo synthesis being 100%; in cases where the fraction of de novo synthesis is between 0 and 100%, the x(15N) of the amino acid should be between the dietary x(15N) and x(15N) of the de novo synthesis pool (±isotope fractionation). When neglecting the relatively small isotope fractionation compared to the high labeling herein, the de novo synthesis fraction as calculated by mass balance was 25% for tyrosine and 40% for proline, despite their near identical x(15N) in consumer. This contrast resulted from differing nitrogen donor pools (tyrosine is supplied by phenylalanine, whereas proline is supplied by the common nitrogen pool, as glutamic acid is the precursor32) that also differed in x(15N) from the common nitrogen pool. Nontransaminating and essential amino acids almost retained their dietary x(15N) value and hence provide highly conserved isotope information from the base of the food web. However, minor changes in isotope compositions might occur (e.g., small deviations from the 1:1 line in Figure 1B) due to isotope fractionation, e.g., caused by the deamination of threonine.13 The half-lives were rather constant among amino acids (Figure 2), indicating that the differences in x(15N) values among amino acids were not influenced by differences in isotope turnover rates that determine the kinetics of label incorporation. This also indicated that the common turnover was not an effect of transamination that differed between amino acids, but rather an effect of proteolysis that governs the degradation of whole proteins and thus affects nearly all amino acids at once. This is not surprising, as transamination is a kinetically perfect reaction, which is so efficient that it is only controlled by the provision of the substrate. Amino acids had shorter half-lives than their bulk muscle tissue,20 although statistically indistinguishable. This might 15

result from the slower turnover of other nitrogen-containing components in bulk muscle, like nucleic acids.33

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +49 (0)8161 713965. Fax: +49 (0)8161 713243. Author Contributions

W.W. designed the feeding experiment, A.V. carried out the feeding experiment, K.A. designed data analysis, A.B. analyzed the data, and A.B and K.A. wrote the manuscript. All authors read and approved the final manuscript. Funding

The “Deutsche Forschungsgemeinschaft” (grant number AU 183/3-1) is thanked for supporting this project. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the valuable discussions with Hans Schnyder on experimental design and with Hanns-Ludwig Schmidt on biochemical pathways. Alan Hopkins provided linguistic support.

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ABBREVIATIONS USED C, stable isotope of carbon with isotopic mass 13 u; δ13C, carbon isotope composition in permil; 15N, stable isotope of nitrogen with isotopic mass 15 u; δ15N, nitrogen isotope composition in permil; x(15N) nitrogen isotope composition in atom fraction; EC, enzyme commission number; SD, standard deviation. 13

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REFERENCES

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Transamination Governs Nitrogen Isotope Heterogeneity of Amino

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