Progress in Earth andPlanetary Science

Ohkouchi et al.
Progress in Earth and Planetary Science(2024) 11:44
https://doi.org/10.1186/s40645-024-00646-7

Open Access

RESEARCH ARTICLE

Regulation of nitrogen isotopic ratiosof cellular components
Naohiko Ohkouchi1* , Yuta Isaji1 and Nanako O. Ogawa1

Abstract
This paper is an attempt to generalize the nitrogen isotope information of the molecules that make up the cellfrom our point of view. Nitrogen in the cell exists as 20 proteinaceous amino acids, nucleobases, hemes, chlorophylls,
and others, and their composition is similar among organisms. Based on a physiologically simple autonomous systemthat maintains a balance between inputs and outputs, it is theoretically predicted that 15N is distributed to eachcellular compound with a certain regularity, and thus a specific relationship in nitrogen isotopic ratios among compounds. Previous studies essentially confirm this. The nitrogen isotopic ratio of compound i constituting the cell
can be generalized as δ15Ni = δ15Nplant + Δi (TP − 1) + γi, where TP represents the trophic position, and Δi and γi are
the trophic discrimination factor and the intracellular 15N distribution of compound i, respectively. Knowing reliablevalues of Δi and γi will help us to better understand nitrogen dynamics in the biosphere and advance our understanding of the picture of the Earth’s environment through biogeochemistry.
Keywords Cell, Nitrogen isotopic ratio, Amino acids, Chlorophylls
1 Introduction
Since nitrogen is an essential element for living organisms, its uptake and metabolism are fundamental information for understanding living organisms. On the other
hand, while stable nitrogen isotopic ratio, a fundamental
tool in geochemistry, has long been employed to study
interactions among organisms in food webs and biogeochemical cycles in natural environment (Wada 1980;
DeNiro and Epstein 1981; Minagawa and Wada 1984;
Altabet and Francois 1994; Sigman et al. 2009), they
have not been used as much to understand the organisms themselves and the cells they comprise. However,recent advances in the study of nitrogen isotope ratios
of individual amino acids make it clear that it is a useful tool for providing deeper insight into these topics.

*Correspondence:
Naohiko Ohkouchi
nohkouchi@jamstec.go.jp
1
Biogeochemistry Research Center, Japan Agency for Marine-Earth
Science and Technology, 2‑15 Natsushima‑cho, Yokosuka, Kanagawa
237‑0061, Japan

Compound-specific nitrogen isotopic ratio of various cellular molecules, including amino acids, which make up
living organisms, is being measured for a variety of purposes and applied to a wide range of studies (e.g., Macko
et al. 1987, Hare et al. 1991, Petzke et al. 2005, Sachs et al.
1999, Chikaraishi et al 2009, Sherwood et al. 2014, Ohkouchi and Takano 2014, McMahon and McCarthy 2015).
In particular, the nitrogen isotopic ratio of individual
amino acids is essential information for understanding the bulk nitrogen isotopic ratio of living organism,
because nitrogen derived from amino acids constitutes
most of the nitrogen that makes up cells in terms ofquantity (Ohkouchi 2023).
Cells, or organisms as aggregates of cells, have mechanisms that autonomously control the amount of nitrogenand other substances that flow through them. Organisms
are designed as autonomous systems that maintain a balance between inputs and outputs, which is inherently
different from systems controlled by first-order kinetics
often found in geologic topics. At the same time, this has
advantages from an isotopic point of view. That is, one
can predict the existence of certain rules in the isotopic© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
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regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of thislicence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/.

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composition of molecules that govern biological processes. The results of many studies conducted to datecertainly suggest this (McClelland and Montoya 2002;
Chikaraishi et al. 2007; McCarthy et al. 2007; Ohkouchi
et al. 2017; Popp et al. 2007). This paper outlines the system in which this autonomous regulation operates andattempts to further develop Ohkouchi (2023) and generalize the isotopic composition of nitrogen components inthe cell.

factor.’ This simple and fundamental equation for deciphering food chains is widely applied (e.g., Wada et al.
1987; Post 2002Newsome et al. 2010) and has given riseto the field of ‘stable isotope ecology’ (e.g., Fry 2006).
A cell or organism (a collection of cells functioning asa single entity with mass M and isotope ratio δ) can beconceptualized as a single box controlled by first-order
dynamics and represented as in Fig. 1a. Mass balance
and isotope balance in this system can be expressed as
the following two simple differential equations under thecondition that 14N >> 15N:

2 Enrichment of heavy isotopes of bioelements
by predation
It has long been known that the enrichment of heavy isotopes of bioelements (e.g., carbon-13, nitrogen-15, and sulfur-34) occurs in the predation process (Miyake and Wada
1967; Peterson and Fry 1987; Fry 2006). Such isotopic phenomenon is generalized by the following equation:
δTP+1 = δTP + ∆(1)

where δ and Δ represent the isotope ratios and theirenrichment (‰) associated with the predation, and thesubscript TP represents trophic position. The trophic
position, or trophic level, is 1 for plants, 2 for herbivores,
3 for animals that eat herbivores, and so on. Dependingon the combination of diets, decimals are possible (e.g.,
an omnivore that consumes equal parts of plants and herbivores would have TP = 2.5). The trophic position serves
as both an indicator of the position in the food chain anda numerical representation of the energy flow order innature. In Eq. (1), the term Δ is sometimes referred to as
the ‘trophic discrimination factor’ or ‘trophic enrichmentdM= fin − fout
dt

(2)

d[Mδ]
= fin δin − fout δoutdt

(3)

By setting the isotope ratio (δout) of the output of thissimple model as (δ–Δ) and assuming that both mass balance and isotope balance hold true, solving these equations yields:δ = δin + ∆(4)Equation (4) represents the same concept as Eq. (1) andcan explain the heavy isotope enrichment associated withpredation mentioned above. In other words, a cell or an
organism can be thought of as a system that discriminates isotopic ratio by Δ.

Fig. 1 Two approaches to understanding the discrimination of nitrogen isotopes in organisms. a Simplified cellular model as a single box regulated
by first-order kinetics. b Autonomously regulated cellular model based on physiological knowledge of nitrogen metabolism (Ohkouchi 2023). ‘AA’
denotes amino acid pool in vivo. Nitrogen isotopic ratios of diet (d), temporal pool of amino acids (AA), protein reservoir (p), and excrement (ex)are denoted by δd, δAA, δp, and δex, respectively. Flows of nitrogen denoted by f and isotope effects by ε. The isotopic ratios of nitrogen transferred
the indicated pathways are given by δd–εo, δp–ε1−, and δAA–ε2

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A mature cell or organism, as dynamic system, is fairly
well balanced, especially in terms of mass. Otherwise,
the body size of the organism would be variable, expanding and contracting. Even in an organism that is not yet
fully mature, input is often only slightly greater than output. Organisms are highly self-regulating systems, andthe control functions powerfully to maintain a state ofdynamic equilibrium.

Furthermore, hemes and chlorophylls are potentially
important as nitrogenous compounds in cells.
Proteins are large molecules composed of amino acidpolymers and typically have molecular weights ranging
from ­103 to ­106 Da, serving as catalysts for biochemical
reactions such as enzymes. In individual cells, thousands totens of thousands of different proteins are packed at concentrations of 2–4 × ­106 per μm3 cell (Milo 2013). Assuminga spherical eukaryotic cell with an average cell size of 30 μm
in diameter and an average protein mass of 4 × ­104 Da(Fig. 2, Brocchieri and Karlin 2005), the total amount ofproteins within this cell would be 1.9–3.7 ng. Assuming acell density of 1 g ­cm−3, that corresponds to 13–27% of thecell weight. Calculated from the average composition ofamino acids widely observed in organisms, there would be
0.28–0.56 ng of protein-derived nitrogen in the above cells.
The compositional balance of the 20 α-amino acids
that make up proteins is remarkably similar across awide range of organisms, from simple bacteria to complex animals like humans. Figure 3 illustrates the weightpercent compositions of hydrolyzable amino acids fromfour oceanic organisms (Cowie and Hedges 1992; Wilson
and Cowey 1985). Glutamic acid, aspartic acid, and lysine
are the most abundant amino acids, often individuallyaccounting for more than 10% of the total amino acids.
Additionally, non-protein amino acids such as ornithine,β-alanine, γ-aminobutyric acid, and nitrogen-containing
compounds like taurine, while present in cells, are generally found in lower quantities compared to the proteinaceous amino acids (e.g., Cowie and Hedges 1992).

3 Nitrogen in the cell
In nature, cells have a great variety of shapes and sizes,
generally ranging from 10 to several hundred micrometers
for eukaryotic cells. Some eukaryotic cells are exceptionally large, on the order of millimeters, while prokaryotic
cells are usually ~1 µm in diameter (e.g., Alberts et al.
2019). Archaea are even smaller, many of which lack somefunctions and often compensate for functional deficits byparasitism, symbiosis with other organisms, or consortium formation with other microbes.
From a microscopic viewpoint, eukaryotic cells are surrounded by a cell membrane and are composed of variousorganelles, including the nucleus, mitochondria, Golgi
apparatus, endoplasmic reticulum, and cytoplasm. Froma chemical perspective, however, the cell can be viewed asa container filled with a variety of compounds, including
proteins and nucleic acids, among others. Focusing particularly on nitrogen, proteins and nucleic acids, which
control the metabolism of living organisms, are not only
functionally important but also quantitatively important.

Fig. 2 Comparison of the nitrogen amount derived from each intracellular component (protein, nucleic acid, heme, chlorophyll, and creatine)of eukaryotic cell, assuming a cell diameter of 30 μm. See details in text

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Fig. 3 Hydrolyzable amino acid compositions as weight % of four types of oceanic organisms drawn from the data in Cowie and Hedges (1992)
and Wilson and Cowey (1985). a Natural phytoplankton (n = 4); three from Dabob Bay and one from Saanich Inlet, b natural zooplankton (n = 3);
two from Dabob Bay and one from Saanich Inlet, c cultured bacteria (n = 3; Vibrio alginolyticus, Bacillus subtilis, and Pseudomonas fluorescens), and dAtlantic salmon (Salmo salar; n = 2). Glx and Asx denote the sums of glutamate and glutamine, and aspartate and asparagine, respectively. n.d.
denotes ‘no data’

Nucleic acids collectively refer to DNA and RNA, andchemically, they are polymers of nucleotides (nucleic acidbase + sugar + phosphate). The composition of the fivenucleic acid bases (adenine, guanine, cytosine, thymine,uracil), which are the building blocks of nucleotides, has
known to be similar across all forms of life since the midtwentieth century (Elson and Chargaff 1955). The total
amount of nucleic acids per single cell is linearly related
to cell volume (Gillooly et al. 2015). Assuming, like above,a spherical eukaryotic cell with a diameter of 30 μm, thecontent of nucleic acids is 50–80 pg in humans and rats,
and even relatively small organisms such as planktonic
crustaceans Daphnia have ~ 20 pg. Since average nitrogenweight ratio in nucleic acids is 9.5%, the nitrogen content
per cell derived from nucleic acids is 0.002–0.08 ng. In
a typical eukaryotic cell, the amount of nitrogen derived
from nucleic acids is more than an order of magnitudeless than that derived from proteins. In smaller prokaryotic cells, however, the relative amount of nucleic acids
is much higher, in some cases reaching 7% by weight(Alberts et al. 2019). Various derivatives of nucleobases

such as ATP and coenzyme A (CoA) are known, but they
are present in very small amounts.
In addition to amino acids and nucleobases, cells contain a variety of nitrogen-containing compounds. Representative examples include chlorophyll, which is involvedin energy harvesting during photosynthesis; heme, which
involved in oxygen transport, storage, and electron transfer and shares the same tetrapyrrole structure; spermine,a polyamine, hexosamines such as glucosamine; and creatine, which is found in vertebrate muscles. For example,
it is known that carbon derived from chlorophyll present
in plant cells (algae) in the ocean averages ~ 2% of cell
carbon (e.g., Cloern et al. 1995). Chlorophyll a (chemical formula: ­C55H72O5N4Mg) contains 6.3% nitrogen byweight, so in a cell of the above size, the average amountof nitrogen derived from chlorophyll is only ~ 0.002 ng,
which is nearly equivalent to the amount of nitrogen
derived from nucleic acids.
Heme is found in over 2300 different proteins (Smithet al. 2010) and serves as cofactors catalyzing various
biochemical reactions. However, even heme B, the mostOhkouchi et al. Progress in Earth and Planetary Science(2024) 11:44

quantitatively important heme, is two orders of magnitude less abundant than chlorophyll by weight in plant
cells (Isaji et al. 2020). Bilirubin and other heme metabolites are also not worth considering in terms of quantity
because they are several orders of magnitude less abundant than amino acids. As an exception, the amount ofphycobilins in cyanobacteria is known to be in range
comparable to that of chlorophylls (Beale 1993).
Creatine, found in vertebrate muscles, contains ~5 gof nitrogen per kg of muscle (Hultman et al. 1996). This
amount is roughly equivalent to some amino acids suchas proline and serine. Thus, creatine is a component thatmust be considered when strictly discussing the nitrogendynamics of vertebrates.
In summary, more than 90% of the nitrogen in eukaryotic cells is derived from the α-amino acids that make up
proteins, while nitrogen in prokaryotic cells is derived
from α-amino acids and nucleobases (Fig. 2). From thispoint on, we will focus primarily on eukaryotic cells.

4 A physiological model of nitrogen isotope
fractionation associated with metabolism
Considering that most of the nitrogen in cells originatesfrom amino acids, the dynamics of nitrogen in the cellshould be modeled from the perspective of amino acidmetabolism. In physiology and biochemistry, the dynamics of amino acids have long been viewed not as in Fig. 1a
but as in Fig. 1b (e.g., Koch 1962; Bender 2012; Ohkouchi2023). That is, proteins taken up through predation are
broken down into individual amino acids in the gut, andinitially enter the amino acid pool. There, they spontaneously mix with the amino acids generated by protein
breakdown, and some of them go to the protein reservoir(in the case of animals, they become part of the muscle),while the rest are excreted outside the cell.
General geochemical systems are governed by firstorder kinetics, whereas as noted above, biological system
is fundamentally about autonomous control of its reservoir size. The differential equations for the size and isotope ratio of the amino acid pool in the metabolic system
are:
d[AA]
= f0 − f1+ + f1− − f2dt

(5)Page 5 of 12

f0 = f2

Since we can assume that the AA pool size is also kept
constant:
f1+ = f1− = f1
(8)

such a balance is indeed widely observed in healthy
adult animals and humans (e.g., Overman and Parrish
2001; Bender 2012).
As is known empirically, nitrogen isotope balance isdalso nearly achieved (i.e., d[AA]δ
= 0). The isotopic fracdt
tionations associated with peptide hydrolysis catalyzed
by protease to individual amino acids (ε0 and ε1−) andprotein synthesis (ε1+) are assumed to be zero for simplicity. In contrast, as some experimental studies haveshown, the amino acid metabolic step involving transamination and deamination described above accompanies
the significant isotopic fractionation (ε2). The isotope
balance Eq. (6) can be arranged to:f1
δd − δAA + ε2=
f0
δAA − δp

(9)

In the cases of investigated healthy higher animals
including human, ff01 ratio for the maintenance of proteins
generally falls in a range 2–3 (Bender 2012; Bergström
et al. 1974; Wagenmakers 1999; Munro 1970; Houlihan
et al. 1993). A significant proportion of the amino acids
released by protein catabolism are reused in the synthesisof new proteins. Since in most animals the size of theprotein pool is 2–3 orders of magnitude larger than thatof the temporal amino acid pool, δp can be regarded as
the bulk isotope ratio of the whole organism (δbulk).
When we apply Eq. (4) to Eq. (9), the magnitude of 15N
enrichment due to predation in organisms can be
expressed by the following equation:
f1∆bulk = ε2 + 1 +(δbulk − δAA )(10)
f0
On the right-hand side of Eq. (10), the first term ε2 canbe considered a constant, while the second term is a variable representing the organism’s
response
to the environ
ment. In the second term, 1 + ff01 is a positive value, but(δbulk–δAA) becomes negative, resulting in the secondd[AA]δd= f0 (δd − ε0 ) − f1+ (δAA − ε1+ ) + f1− (δp − ε1− ) − f2 (δAA − ε2 )dt
where f terms are fluxes and (δ–ε) terms are isotopic
ratios for fluxes. In mature animals, inputs and outputs
are in balance, so the following equation holds.

(7)

(6)

term on the right side as a whole is negative. In otherwords, Δbulk value (i.e., 3.4‰ on average) is smaller than ε2and dynamically determined (Fig. 4). However, since theOhkouchi et al. Progress in Earth and Planetary Science(2024) 11:44

Fig. 4 Schematic illustration of Eq. (10) regarding Δbulk shownin the upper panel. In the lower panel, observation results arealso shown as a frequency distribution of Δbulk values (0.5‰increment; redrawn from Post 2002). Broken line indicates mean Δbulk
value (3.4‰)

cell is an autonomously regulated system, the Δbulk value
falls within a relatively narrow range and has been empirically considered as a constant in ecological applications.
For example, one would expect that starvation (f0 → 0)
would increase the value of the second term on the righthand side (1 + f1/f0) and also increase the isotopic ratio of
amino acid pool (δAA), which strongly reflect fractionation
during amino acid degradation (ε2), leading to a decreasein the value of Δbulk. However, as critically evaluated by
Doi et al. (2017), observations do not necessarily replicate
this prediction, and the nitrogen isotopic ratio does notchange significantly even after experiencing starvation.
This may be explained by a metabolic switch in responseto changes in the external environment, such as starvation, that reduces f1 and f2 (the latter in particular has theeffect of reducing δAA) and kept the Δbulk value constant.

5 Mechanism of 15N enrichment
As described earlier, since most of the nitrogen in the cellis derived from proteinogenic amino acids, the nitrogenisotopic ratio of the bulk organism should approximately
correspond to the weighted average of the isotopic
ratios of its 20 amino acids. Focusing on the dynamics ofPage 6 of 12

nitrogen (amino group) of amino acids, the main metabolic processes are aminotransfer reaction and degradation by dehydrogenase reaction (oxidative deamination)(Fig. 5).
The aminotransfer reactions are responsible for the
redistribution of 15N between each amino acid in thecell. They are reactions in which amino groups of various
amino acids are transferred to 2-oxoacids (α-ketoacids)
to form different α-amino acids. Pyridoxal 5’-phosphate(PLP) functions as a cofactor in the active center of various aminotransferases that catalyze amino group transfers (Eliot and Kirsch 2004; Ohkouchi et al. 2015, Fig. 5a).
In other words, once the amino acid binds to PLP toform a complex, it dissociates the 2-oxoacid after several
intermediates from the complex. The amino group, via a
temporary binding to PLP, is soon transferred to another
2-oxoacid to form a new amino acid. Most of the aminogroups are eventually transferred to 2-oxoglutaric acid toform glutamic acid.
In such amino group transfer reactions, the following
two points are important when considering the nitrogenisotopic ratio. First, it is the amino acid with a deprotonated amino group (R-NH2) in which the amino group
has a lone electron pair, that forms a complex with PLP.
When amino acids with deprotonated amino groups arein isotopic equilibrium with their protonated (R-NH3+)
counterparts in the cell, their isotopic fractionation
exceeds 16‰ (Hermes et al. 1985; Rishavy and Cleland
2000; Oi et al. 2023). Given the neutral to somewhat alkaline pH of the cells, the majority of amino acid aminogroups would be in a protonated state, while the δ15N
values of the minor deprotonated amino acids would bemuch lower than the protonated ones.
Another isotope fractionation occurs at the Schiff baseformation between the deprotonated amino acids and
PLP (Rishavy and Cleland 2000). The association anddissociation of the deprotonated amino acids with PLP
is reversible, with preferential dissociation of R-14NH2
from the PLP. Thus, when the subsequent reaction stepis rate-limiting, R-15NH2 complexed with PLP would
preferentially proceed to the final product. Histidine
and glutamate carboxylases, whose reactions are ratelimited at decarboxylation step that occur immediately
after the Schiff base formation, have shown 15N enrichments in the products up to 23‰ (Abell and O’Leary
1988). In case of aminotransferases, this 15N enrichment
partially offsets the equilibrium 15N-depletion in the substrate R-NH2, leading to formation of other amino acids
depleted in 15N by up to ~ 8‰ (Macko et al. 1987; Rishavy
and Cleland 2000; Goto et al. 2018).
In contrast, in oxidative deamination, the amino groupsof each of the 20 amino acids are first assembled into
glutamate by an aminotransfer reaction. The glutamate

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Fig. 5 Overviews of chemical mechanisms of cellular reactions related to amino group of amino acids. a A half-reaction of transamination(deamination) of amino acids catalyzed by aminotransferase. PLP indicates pyridoxal 5′-phosphate, common cofactor of aminotransferases,
and PMP pyridoxamine 5′-phosphate. b Oxidative deamination of glutamic acid catalyzed by glutamate dehydrogenase (GDH). In the cell, thisreaction proceeds primarily from left to right. E is enzyme and broken lines indicate nucleophilic attacks

is then decomposed into 2-oxoglutarate and ammonium
by glutamate dehydrogenase (GDH) in the mitochondria(Fig. 5b). This reaction is reversible, but because of the
low affinity of ammonium for GDH, glutamate is usually deaminated to yield ammonium. The ammonium isconverted to urea through the urea cycle and excreted
from the body. Thus, the metabolic enrichment of 15N
is mainly due to the oxidative deamination of glutamate,which produces 15N-depleted ammonium that is thenremoved from the body. If the oxidative deamination by
GDH is in equilibrium, 15N is enriched by 47‰ on theglutamate (Schimerlik et al. 1975; Cleland 1982). This
15
N enrichment is partially offset by the effect of the aminotransfer reaction described earlier, which produces
15
N-poor glutamate.

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6 Generalization of nitrogen isotopic ratios
of intracellular compounds
The composition of the 20 amino acids that make up
proteins is relatively similar, even among very different organisms such as microbes and higher organisms,
or organisms at different trophic positions (Fig. 3, Ohkouchi 2023). This implies that the f1/f0 ratios of individual
amino acids are comparable among different organisms,
as described above, suggesting that the discussions mentioned earlier at the bulk level are essentially true at theindividual amino acid level. Since most of the nitrogencontained in eukaryotic cells originates from the aminoacids that make up proteins, we go one step further to discuss the nitrogen isotope ratios of individual amino acids.
Here, let us introduce the concept of ‘isotope distribution within the cell (γ)’ as a perspective to consider
the isotope ratios of various compounds synthesizedwithin the cell. The γ value is defined as the deviationof compound i from the bulk isotope ratio of the cell(γi ≡ δ15Ni − δ15Nbulk). With this concept, we can generalize the nitrogen isotopic ratio of compound i, whichconstitutes the cell (i.e., naturally occurring compounds),
using the following equation:

Table 1 Current summary of intracellular 15N distribution (γ, ‰)in the cell and trophic discrimination factor (Δ, ‰) for aquaticorganisms based primarily on our previous studies. These values
can be applied to Eq. (11) in the textδ 15 Ni = δ 15 Nplant + ∆i (TP − 1) + γi

(11)

On the right-hand side of Eq. (11), the first term is theecosystem-base term, the second term can be called thetrophic term, and the final term, γi, represents the intracellular isotope distribution to compound i, as describedabove. Table 1 and Fig. 6 summarize the current versionof Δi and γi values for aquatic organisms, based primarily on our previous studies. These figures are still controversial for some compounds and should be updated in
the future (e.g., McMahon and McCarthy 2016). As with
the Δbulk value discussed above, at least the Δi values are
inherently variable, albeit within a relatively narrow range(with some exceptions). And if metabolic flux changes,
for example due to dietary imbalances over time, thisshould be accompanied by a change in the Δi value (Chikaraishi et al. 2015). With this in mind, the values shownin Table 1 need to be validated and discussed.
Studies of nearly 20 nitrogenous compounds fromvarious aquatic organisms have revealed a wide rangeof γi values from − 7 to + 11‰ for each compoundconstituting the cell (Table 1). In particular, chlorophyll a from freshwater cyanobacteria has the highest
γi values, indicating the existence of an intracellular
15
N enrichment mechanism (Katase and Wada 1990;
Sachs et al. 1999; Higgins et al. 2011, Ohkouchi et al.
2006). In aquatic organisms, there is little differencein the γi value of each amino acid between organisms
from different evolutionary lineages, such as algae andCompound

γ (‰)

Δ (‰)

References

Amino acid
Glycine–4.9 ± 3.6

3.7 ± 3.9

1

Alanine0.6 ± 1.8

6.1 ± 2.1

1

Serine–7.2 ± 2.6

3.6 ± 3.0

1

Aspartic acid*~ 1**

3.4 ± 1.8

2

Threonine~ 2**–6.6 ± 3.4

2

Valine2.0 ± 1.8

5.0 ± 1.7

1Glutamic acid***0.8 ± 1.6

8.0 ± 1.2

1Methionine–4.6 ± 1.4

0.5 ± 0.6

1

Proline0.5 ± 2.1

6.1 ± 1.6

1

Leucine–0.3 ± 2.1

4.8 ± 2.0

1Isoleucine

0.3 ± 1.5

4.8 ± 1.7

1

Lysine~ –6**–1.3 ± 0.9

2

Phenylalanine–2.6 ± 1.3

0.4 ± 0.5

1

Tyrosine–8 to –6

n.d.

2

Phytoplankton

~0

n.d.

3

Freshwater cyanobacteria

11.4 ± 5.1

n.d.

3

Heme B

Chlorophyll a
Marine algae–4.7 ± 1.7–

4, 5, 6

Marine cyanobacteria

1.3 ± 2.3–

4, 5, 6

Freshwater cyanobacteria

11.0 ± 2.6–

4, 5, 6, 7

Divinylchlorophyll a

1.0 ± 1.3–

8

*Combined with asparagine**No data reported in the reference, estimated from the figure***Combined with glutamine
Error indicates 1σ
n.d.: not determined
References: 1: Chikaraishi et al. (2009), 2: McMahon and McCarthy (2016), 3:
Isaji et al. (2020), 4: Sachs et al. (1999), 5: Ohkouchi et al. (2006), 6: Higgins et al.
(2011), 7: Katase and Wada (1990), 8: Isaji et al. (2020)

cyanobacteria (Chikaraishi et al. 2009), but there areclear differences in chlorophylls. This suggests thatsomewhere in the process of chlorophyll synthesis fromprecursor amino acids (i.e., glutamic acid and glycine)is the source of the difference in isotopic ratios, andthat the process(es) probably emerged during evolution.
On the other hand, the Δi values of amino acids show
a distribution of more than 15‰ from − 8 to + 8‰.
Some amino acids, such as phenylalanine and methionine, show little change in nitrogen isotopic ratio withpredation processes (i.e., metabolism). These aminoacids are called ‘source amino acids’ and are considered to have a Δi value close to zero because protonation–deprotonation and aminotransfer reactions are
not directly involved in their metabolism. In fact, inOhkouchi et al. Progress in Earth and Planetary Science(2024) 11:44

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Fig. 6 A cross-plot of intracellular 15N distribution (γ) and trophic discrimination factor (Δ) for compounds presented in Table 1. Open symbols
indicate amino acids for which the error in the γ value is unknown (i.e., asparatate, threonine, and lysine). Error bars indicate 1σ

metabolism, phenylalanine is hydroxylated to form
tyrosine, and methionine is catalyzed by ATP to form
S-adenosylmethionine. The metabolisms of threoninewith a negative Δi value and lysine with a relatively
small Δi value are not also involved in aminotransfer
reactions.

7 Estimation of trophic position
Equation (11) demonstrates that if the Δi and γi values
for each compound are known in advance, the nitrogenisotope ratio of primary metabolic products synthesized by organisms will become a function of the δ15N
value of the ecosystem pool to which the sample organism belongs (the ecosystem-base term) and its trophic
position. Now considering compounds ‘a’ and ‘b’ within
the same organism and taking the difference of Eq. (11)for them, we obtain,

δ 15 Na − δ 15 Nb = (∆a − ∆b )(TP − 1) + (γa − γb )(12)
and the ecosystem-base term is eliminated. Solving thisequation for TP:
TP = (δ 15 Na − δ 15 Nb − (γa − γb ))/(∆a − ∆b ) + 1(13)This yields a general formula to determine the trophic
position.
Substituting two amino acids with relatively small error
γi and Δi values into Eq. (13) yields a TP with relatively
small error. Typical examples of such amino acids areglutamic acid (Glu) and phenylalanine (Phe), which havevery different Δi values (Table 1). Equation (14) can bederived for aquatic organisms (Chikaraishi et al. 2009):
TP = (δ 15 NGlu − δ 15 NPhe − 3.4)/7.6 + 1(14)

Ohkouchi et al. Progress in Earth and Planetary Science(2024) 11:44

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In other words, by measuring the nitrogen isotope
ratios of the two compounds, glutamic acid and phenylalanine, in a sample organism, it is possible to estimate
the trophic position of that organism. To accurately estimate TP, (γa–γb) and (Δa–Δb) must not change much. In
fact, evidence has been presented and discussed for various aquatic plants and prey–predator combinations as tohow much these values vary (e.g., Chikaraishi et al. 2009;
McCarthy et al. 2013; McMahon and McCarthy 2016;
Decima et al. 2017; Whiteman et al. 2019). If TP is calculated using the combination of glutamate and phenylalanine, the estimation error is generally ~ 0.1.
While the variability of amino acid nitrogen isotope ratios has been discussed, the TP obtained by thismethod has provided us with a variety of information,such as the feeding habits of organisms that are difficult
to observe (e.g., deep-sea organisms) and basic information on fishes useful in fisheries (Miller et al. 2012,
Ohkouchi et al. 2013, Tsuchiya et al. submitted). Furthermore, the TP information has also been applied todiverse studies, including the efficiency of energy transfer
through trophic pathways, the effect of eutrophication onfish diet, and paleoanthropogenic diet analysis (e.g., Ishikawa et al. 2022; Ogawa et al. 2013; Naito et al. 2016).

The greatest advantage of applying amino acid nitrogenisotope ratios to natural biological samples is the ability
to separate the two inseparable pieces of information in
the bulk nitrogen isotope ratios, TP and ecosystem-baseδ15N. The importance of separating these has often been
overlooked. One of the important applications of ecosystem-base δ15N is its use in isoscape, which has developed remarkably in recent years (Bowen 2010). A notable
example is Matsubayashi et al. (2020), who revealed themigration path of chum salmon (Oncorhynchus keta) in
the North Pacific based on the nitrogen isotope ratio of
phenylalanine contained in the growth ring of vertebral
centra. In addition to vertebral centra, otoliths and eyeballs have been known to accumulate substances by living organisms, and the application of nitrogen isotope
ratios of eyeballs, which are enriched in amino acids,
to isoscape will be developed in the future (McMahon
and Newsome 2019; Harada et al. 2022; Yoshikawa et al.
2024).

8 Estimation of ecosystem‑base δ15N
If TP can be estimated, the other unknown in Eq. (11),δ15Nplant, or ecosystem-base δ15N can be estimated by thefollowing equation:
δ 15 Nplant = δ 15 Ni − ∆i (TP − 1) − γi(15)For example, if applying phenylalanine to Eq. (15):δ 15 Nplant = δ 15 NPhe −∆Phe (TP − 1)− γPhe

(16)

Considering the very small Δi value of phenylalanine(Table 1), a source amino acid, it can be simplified asfollows.

δ 15 Nplant ≃ δ 15 NPhe − γPhe

(17)

Furthermore, the equation becomes more robust if
applying chlorophyll a, which is synthesized only byplants (TP = 1):

δ 15 Nplant = δ 15 Nchl − γchl(18)

If the sample is a mixture or contains a significant
amount of microorganisms, Eq. (18) may be more accurate than Eq. (17). In particular, since chlorophyll nuclei
can persist as porphyrins in sediments for billions ofyears, ecosystem-base δ15N information is useful forreconstructing the nitrogen cycle in the surface ocean
over geologic time.

9 Conclusions and implications
It is often difficult to describe quantitatively the macroscopic phenomena of the Earth’s environment, where
biological processes dominate. Part of this difficulty is
due to the ‘biological opportunism’ with which organisms
change the way they cope with the external environment(in order not to change themselves). This paper proposeda perspective that incorporates autonomous biologicalsystem a priori to solve this problem, and described theintracellular distribution of nitrogen isotopes of mainly
amino acids. The nitrogen isotopic balance among thevarious nitrogeneous compounds in the biosphere shouldbe essentially explained by γi and Δi values, the isotopic
discrimination associated with biological nitrogen uptake
and assimilation. With more robust information on γi andΔi, we will have a better understanding of the nitrogendynamics in the biosphere and further progress in understanding the whole picture of the Earth’s environment
through biogeochemistry.
The same should, of course, be true for other isotopesof bioelements such as carbon, as described earlier.
Decarboxylation of amino acids, like deamination, is a
PLP-dependent reaction that involves fractionation ofcarbon isotopic ratio (McCarthy et al. 2004; Takizawa
et al. 2020; Sun et al. 2020). However, since C–C bondis stronger than C–N bond and it form the major backbone of all biological compounds, carbon isotopic ratios
should not vary as much as nitrogen isotope ratios, which
has been often confirmed in nature (e.g., Wada 2009; Fry
2006). Furthermore, the discussion developed in thispaper is not limited to the Earth. If an autonomous system (maybe life?) exists somewhere off Earth, we wouldfind a specific relationship between the isotopic ratios ofOhkouchi et al. Progress in Earth and Planetary Science(2024) 11:44

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each component. The regularity of isotope ratios predicts
the system that controls them.

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Abbreviations
Glu	Glutamate
Phe	Phenylalanine
Chl	Chlorophyll
Acknowledgements
We thank Eitaro Wada, Yoshito Chikaraishi, Yoshinori Takano, Naoto F. Ishikawa,and Chisato Yoshikawa for discussion that forms the basis of this paper, and T.
Okochi for advices in preparing Fig. 5. We also thank two anonymous reviewers for improving the manuscript.
Author contributions
NO proposed the topic, and conceived and designed the study. YI and NOO
analyzed the data and helped in their interpretation. All authors read andapproved the final manuscript.
Funding
This work was supported by JSPS KAKENHI Grant Number 20H00208 and
23H0015.
Availability of data and materials
Data sharing not applicable to this article as no datasets were generated oranalyzed during the current study. Please contact author for data requests.

Declarations
Competing interests
The authors declare that they have no competing interest.
Received: 14 April 2024 Accepted: 16 July 2024

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