Effect of Organic Food Intake on Nitrogen Stable Isotopes

Abstract

Food choices affect the isotopic composition of the body with each food item leaving its distinct isotopic imprint. The common view is that the natural abundance of the stable isotopes of nitrogen (expressed as δ¹⁵N) is higher in animals than in plants that constitute our contemporary diets. Higher δ¹⁵N is thus increasingly viewed as a biomarker for meat and fish intake. Here we show that organic compared to conventional farming increases plant δ¹⁵N to an extent that can appreciably impact the performance of δ¹⁵N as a biomarker. The error that can arise when organic plants are consumed was modelled for the entire range of proportions of plant versus animal protein intake, and accounting for various intakes of organic and conventionally grown crops. This mass balance model allows the interpretation of differences in δ¹⁵N in light of organic food consumption. Our approach shows that the relationship between δ¹⁵N and meat and fish intake is highly contextual and susceptible to variation at the population, community or group level. We recommend that fertilization practices and organic plant consumption must not be overlooked when using δ¹⁵N as a biomarker for meat and fish intake or to assess compliance to nutritional interventions.

1. Introduction

Biomarkers for the intake of plant and animal proteins can help us to understand relationships between diet and health, and to assess adherence to nutritional interventions aiming at modulating reliance on animal and plant proteins. The natural abundance of the stable isotopes of nitrogen (δ¹⁵N) is increasingly viewed as a biomarker for meat and fish intake [1,2,3,4,5]. Recent controlled feeding studies found associations between tissue δ¹⁵N and meat and fish intake [1,2], reinforcing the potential of δ¹⁵N as a dietary intake biomarker.

The rationale behind this approach lies on the fact that organisms are enriched in ¹⁵N relative to their diet [6] likely due to isotopic fractionation during certain metabolic reactions [7]. Animals are, thus, enriched in ¹⁵N compared to the plants and other animals that they consume, an isotopic offset that varies with changes in protein and amino acid metabolism [8,9]. Due to the greater length of aquatic food chains, marine products and fish, particularly when not farm-raised [10], have the highest ¹⁵N content [11]. δ¹⁵N is, therefore, a biomarker of meat and fish intake under the premise that the animal proteins consumed have a higher ¹⁵N content than the plant proteins consumed. The ¹⁵N content depends on the nitrogen cycle from which a product originates with nitrogen conversion processes fractionating isotopes to different degrees depending on the context [12,13,14]. Human activities and agricultural practices affect the nitrogen cycle.

This higher ¹⁵N isotopic abundance in animals than in plants is, indeed, expected in nature. Human mastering of agriculture might have strengthened this difference with the use of synthetic fertilizers. Synthetic fertilizers, an important nitrogen source in modern agriculture, have a low ¹⁵N content (δ¹⁵N close to 0 ‰) because their nitrogen is derived from atmospheric N₂ and fractionation tends to be minimal during their production [12,15]. However, in recent years, the market for organic food has been increasing and land use for organic agriculture expanding [16]. Synthetic fertilizers are prohibited in organic farming, where fertilization is achieved with the application of natural products such as animal manures. According to Bateman and Kelly [13], fertilizers that may be permitted in organic agriculture have a higher ¹⁵N content (mean δ¹⁵N of 8.5 ‰). Many studies have consequently shown a higher δ¹⁵N in organically compared to conventionally grown crops [14] suggesting that the δ¹⁵N of the animal proteins compared to the plant proteins consumed, may not be as high when organic plants are the preferred choice.

This study assesses the impact that organic food and fertilization practices can have on the performance of δ¹⁵N as a biomarker. A nitrogen mass balance model was developed to test the effect of organic plant intake on body proteins δ¹⁵N at various relative intakes of plant and animal proteins. Various scenarios were identified where the performance of δ¹⁵N as a biomarker for fish and meat intake is impacted differently.

2. Materials and Methods

2.1. ¹⁵N Natural Isotopic Abundance of Organic Plants

δ¹⁵N values of edible plants detailed in Supplementary Materials Table S1 were obtained from published literature. As there are more published δ¹⁵N data for conventionally grown plants, only studies containing data for both crops considered as organic and conventionally grown were included in our analysis to avoid having imbalanced data in terms not only of plants categories per farming method, but also of sample size. This resulted in the inclusion of 21 studies in our analysis (with 80 and 90 δ¹⁵N values for conventionally and organically grown crops, respectively). The isotopic offset between organic and conventionally grown plants (Δ¹⁵N_org-conv) was determined from the median δ¹⁵N values of the two farming methods:

Δ¹⁵N_org-conv = δ¹⁵N_{organic plants} − δ¹⁵N_{conventional plants}

(1)

2.2. Effect of Organic Plant Protein Intake on Body ¹⁵N Natural Isotopic Abundance

A two-sources isotope mixing model can be use to relate the proportions of plant to animal proteins consumed to tissue δ¹⁵N if the isotopic offset between tissue and dietary proteins owing to metabolism is considered. Human tissues δ¹⁵N equals the sum of the δ¹⁵N of the different proteins consumed, weighted by their proportion to dietary nitrogen, plus an isotopic discrimination factor that is linked to amino acid and protein metabolism [17,18]. As detailed in Supplementary Materials Text S1, separating the nitrogen consumed into nitrogen from either animal or plant sources and further separating the plant sources into conventionally and organically grown plants, the impact of organic food intake on tissue protein δ¹⁵N can be assessed using a simple isotopic mass balance calculation:

δ¹⁵N_{tissue proteins} = δ¹⁵N_animal × P_animal + (δ¹⁵N_{conv plants} + Δ¹⁵N_org-conv × P_org) × (1-P_animal) + Δ¹⁵N_tissue-diet

(2)

where δ¹⁵N_{tissue proteins} is the isotopic composition of the body proteins, δ¹⁵N_{conv plants} and δ¹⁵N_animal are the δ¹⁵N values of dietary plant and animal proteins, respectively. P_animal is the proportion of dietary proteins occupied by animal proteins and 1-P_animal is the proportion occupied by plant proteins (where protein refers to protein nitrogen). P_org is the proportion of dietary plant proteins occupied by proteins from organically grown plants. Δ¹⁵N_tissue-diet is the isotopic change between tissue and dietary proteins owing to metabolism, set here at 3.5‰ (an approximation based on the liver proteins analyzed after the consumption of two diets with different effects on amino acid metabolism [17]).

The error resulting from organic plant intake on δ¹⁵N_{tissue proteins} was then calculated as follows:

δ¹⁵N absolute error = Δ¹⁵N _org-conv × P_org × (1-P_animal)

(3)

δ¹⁵N relative error = 100 × δ¹⁵N absolute error/[δ¹⁵N_animal × P_animal + δ¹⁵N_{conv plants} × (1-P_animal) + Δ¹⁵N_tissue-diet]

(4)

δ¹⁵N_{tissue proteins} and its absolute and relative error were computed over the whole range of P_animal (0 to 1) and for each 0.1 (10%) increment in P_org. Calculations were performed over the whole range of δ¹⁵N values observed in animal proteins according to Huelsemann et al. [11], with the lowest end of δ¹⁵N_animal at 2,83‰, the average δ¹⁵N of poultry, and the highest end of δ¹⁵N_animal at 12.39‰, the average δ¹⁵N of fish. As in Equation (1), δ¹⁵N_{conv plants} was set as the median of the literature values for conventionally grown plants.

3. Results

3.1. ¹⁵N Natural Isotopic Abundance of Organic Plants

Literature δ¹⁵N data for plants grown conventionally or using organic fertilization are displayed in Figure 1. Crops fertilized organically tended to have a higher δ¹⁵N. The mean δ¹⁵N of organically grown plants was 7.7 ± 4.4‰ (median = 7.2‰) while it was 2.8 ±2.3‰ (median = 3.0‰) for plants fertilized inorganically resulting in a Δ¹⁵N_org-conv of 4.2‰. Figure 1 shows an overlap between the δ¹⁵N values of organically and conventionally grown plants. The lowest δ¹⁵N value for conventional farming was observed in a batch of tomatoes and was of −2.5‰ [15] while the highest value, 8.72‰, was observed in peppers [19] (Table S1). For organic agriculture, the lowest δ¹⁵N value, 0.3‰, was measured in peas obtained from an organic grocery store [20] and the highest δ¹⁵N values, 21.89‰, in lettuce fertilized with bat guano [21] (Table S1).

3.2. Effect of Organic Plant Protein Intake on Body ¹⁵N Isotopic Abundance

Model output (δ¹⁵N_{tissue proteins}, Equation (2)) is shown in Supplementary Materials Figure S1. At the low end of the δ¹⁵N_animal spectrum, δ¹⁵N_{tissue proteins} increases not only with organic plant intake (Figure S1A), but also with plant protein intake. The opposite relationship between δ¹⁵N_{tissue proteins} and plant protein intake is seen at the high end of the δ¹⁵N_animal spectrum (Figure S1B).

δ¹⁵N relative error introduced by organic plant consumption is plotted in Figure 2 as a function of the contribution of plant proteins to total protein intake (1-P_animal). Figure S2 shows the absolute error. As seen from Equation (3), the absolute error is independent of δ¹⁵N_animal. At the usual level of plant protein intake observed in the French and American populations (~30% of protein from plant sources, 1-P_animal = 0.3; [22,23,24]), the error ranged from 0.1‰ to 1.3‰ for low to high P_org (0.1 to 1).

Figure 2 shows the relative error for both the low (Figure 2A) and high (Figure 2B) ends of the δ¹⁵N_animal spectrum. When δ¹⁵N_animal of the dietary intake is low, a situation where the major source of animal protein is poultry, δ¹⁵N relative error seems to vary linearly with the relative importance of plant proteins to total protein intake (Figure 2A). In this situation, at the usual level of plant protein intake in the French and American populations (~30% of protein from plant sources, 1-P_animal = 0.3; [22,23,24]), δ¹⁵N relative error ranged from 2.0% to 19.8% for low to high P_org. In contrast, Figure 2B shows that when δ¹⁵N_animal of the dietary intake is high, a situation where the main source of animal protein is fish or marine products, δ¹⁵N relative error vary non-linearly with the relative importance of plant proteins to total protein intake. In this case, at the usual level of plant protein intake, δ¹⁵N relative error ranged from 1.0% to 9.6% for low to high P_org. Maximal error occurs when all of the plant protein intake is from organic sources (P_org = 1), and the proportional plant protein intake is high (1-P_animal close to 1). In that context, for both the low (Figure 2A) and high (Figure 2B) ends the δ¹⁵N_animal spectrum, the maximal δ¹⁵N absolute error is 4.2‰ and the relative error 64.6%. As shown in Equation (4), relative error was not only a function of δ¹⁵N_animal, but also dependent on Δ¹⁵N_tissue-diet.

4. Discussion

Whereas natural ¹⁵N abundance is a candidate biomarker for meat and fish intake [1,2] due to a higher ¹⁵N content in meat and fish than in plants, it has been shown that organic compared to conventional farming increases plant δ¹⁵N [14]. The present study demonstrates that this isotopic particularity of organic plants impacts the performance of δ¹⁵N as a biomarker in humans, for meat and fish intake. Proportional changes in organic compared to non-organic plant consumption impact the body δ¹⁵N differently depending on plant and animal protein intakes, and on the δ¹⁵N of the animal proteins consumed.

4.1. Organic Farming Impacts Plant Nitrogen Isotopic Composition

Plant δ¹⁵N was lower on average for conventionally grown plants than for organically grown plants (Δ¹⁵N_org-conv = 4.2‰, Figure 1) due to the use of fertilizers with a high ¹⁵N content such as animal manures in organic agriculture. Huelsemann et al. [11] reported a similar yet slightly lower average difference of 3.7‰ between vegetables grown conventionally versus organically with a δ¹⁵N of 6.8‰ in organic plants. In the present study, organic plants had an average δ¹⁵N of 7.7‰, a value higher than the average δ¹⁵N of many animal products. It must be emphasized that these average isotopic differences between conventionally and organically grown plants are highly contextual and dependent on the nitrogen cycle from which the products originate. It has been shown, for instance, that organic fertilization with green manure alone instead of animal manure can result in lower plant δ¹⁵N [25,26]. Local practices and potentially rapidly evolving national regulations on organic fertilization must be taken into account when assessing the impact of organic farming on plant δ¹⁵N, and thus on body δ¹⁵N as a biomarker for fish and meat intake in a population, or a community, and as a marker of compliance in study participants.

4.2. Organic Plant Intake Affects the Performance of δ¹⁵N as a Biomarker

We demonstrate here that organic plant intake can affect the performance of ¹⁵N natural isotopic abundance as a biomarker for meat and fish intake. This idea was illustrated by computing δ¹⁵N changes introduced by organic plant consumption using a mass balance model accounting for proportional intakes of plant and animal proteins (Figure 2). Various proportional intakes of organic relative to non-organic plants were also considered to simulate different frequencies of purchasing organic foods. The goal of this approach was, rather than to reflect the average behaviour of a population, to explore all possibilities that could be seen among individuals and between different populations and communities.

More precisely, potential behavioural differences between individuals affecting their tissue δ¹⁵N that were studied were: (1), the intake of plant relative to animal proteins (1-P_animal), (2) the intake of organic relative to non-organic plant proteins (P_org), and (3) the δ¹⁵N of animal protein intake (Figure 2A vs. Figure 2B), which depends on the types of animals consumed.

Figure 2 shows that the impact of organic plant consumption on tissue δ¹⁵N depends on the intake of plant relative to animal proteins. Studies of the French and American populations showed that plant proteins represent, on average, around 30% of the proteins consumed (1-P_animal = 0.3) [22,23,24]. The proportion of plant protein intake is at 100% on a vegan diet, and when organic plants are consumed, this has the greatest impact on tissue δ¹⁵N.

Although the effect of organic farming on the δ¹⁵N of vegetables was undeniable in a previous study, it was argued that organic plant intake has a minor effect on the body δ¹⁵N of contemporary Germans, who obtains few of their proteins from vegetables and around 30% from cereals, as no significant difference between conventionally and organically grown cereals were observed [11]. There is, however, data clearly showing that organic fertilization can impact cereals δ¹⁵N [25,26,27], indicating that such a generalization may not be possible.

We acknowledge some limitations of the current approach. The first one lies in the estimation of Δ¹⁵N_org-conv since very little data are available for some plant categories like legumes that tend to have a low δ¹⁵N due to atmospheric N₂ fixation. A smaller or minor effect of organic fertilization could thus be expected in these plants. Nonetheless, legumes account for a small proportion of total plant protein intake in some populations [24]. This shows that Δ¹⁵N_org-conv and, therefore, the effect of organic plant intake on δ¹⁵N, is highly circumstantial and dependent on the nitrogen cycle from where consumed plants are grown. Furthermore, the lower digestibility of some vegetable proteins was not explicitly accounted for in Equation (2), which means that proportional plant protein intake (1-P_animal) as used here is the contribution of effectively digested plant proteins to total effectively digested proteins.

4.3. Animal Protein Sources Affect the Performance of δ¹⁵N as a Biomarker

The source of animal protein is another modulator of the performance of δ¹⁵N as a biomarker that was studied. There is a well-documented δ¹⁵N spectrum between different animal categories with poultry at the lower end and fish at the higher end of the δ¹⁵N values and with considerable δ¹⁵N variability within the animal categories [10,11]. While consumers that choose organic plants might also prefer organic meat, there appear to be no clear effects of organic animal production on their δ¹⁵N, which must also be considered as highly context dependent [28]. At the two ends of this δ¹⁵N spectrum of animal products, δ¹⁵N behave in opposing ways with changes in organic plant intake. When organic plants are consumed and poultry is the main source of animal proteins, increasing proportional intake of plant proteins makes the body δ¹⁵N closer to that of meat and fish, while it is the opposite when animal proteins are predominantly from an aquatic origin (Figure S1). This reemphasizes that the capacity of δ¹⁵N as biomarker for meat and fish intake [1,2] is very contextual. Furthermore, these opposite δ¹⁵N behaviors between the two modeled dietary scenarios (Figure S1) show that the directionality of the error that ranges between 0‰ and Δ¹⁵N_org-conv (Figure S2, Equation (3)) depends on the difference in δ¹⁵N between dietary plants and animals. If the δ¹⁵N is higher in dietary plants than in animal proteins, the error is positive; when δ¹⁵N of animal proteins is higher the error is negative. Opposite error directionalities show that the common view that meat intake increases tissue δ¹⁵N is not always valid. Although, as can be seen from Equation (3), the absolute isotopic impact in ‰ of organic plant intake (Figure S2) is independent of the source of animal proteins, δ¹⁵N relative error propagates differently along the plant protein intake axis depending on the δ¹⁵N of the animal proteins consumed (Figure 2). Having poultry as the main source of animal proteins results in a higher relative impact at low proportional intake of plant proteins (Figure 2A). In contrast, if the main source of animal proteins is from an aquatic origin, the relative impact on δ¹⁵N of organic plant intake will increase more sharply when the proportional intake of plant proteins is high (non-linear increase in Figure 2B). This latter scenario is of relevance to pescatarians on a predominantly plant-based diet supplemented with a small amount of fish.

Different relative error propagations due to varying sources of animal proteins raises the question of the meaning of the relative error for an isotopic biomarker. According to our model predictions, this might depend on the δ¹⁵N of the studied population and on its homogeneity in terms of δ¹⁵N. Relative error could reflect the effect size of the relationship between animal protein intake and δ¹⁵N, which can differ across populations due to different δ¹⁵N values of the animals and plants consumed. This should be explored more deeply in future work and pertains to another caveat of our approach which is that the isotopic offset between tissue and dietary proteins (Δ¹⁵N_tissue-diet in Equation (2)) is likely to increase with plant protein intake [17] due to changes in amino acid metabolism. In proportion, metabolically induced δ¹⁵N variations have different impacts depending on the δ¹⁵N of consumed proteins.

5. Conclusions

Natural differences in nitrogen isotopic abundance between individuals, is increasingly viewed as a biomarker for meat and fish intake. With an isotopic mass balance equation, considering proportional animal and plant protein intakes, we show that this is not strictly true when organic plants are consumed. There can even be opposite relationships between tissue ¹⁵N content and animal protein intake depending on the context. There is thus no systematic relationship between meat and fish intake and human tissue isotopic composition. Both fertilization practices and their potential evolution, and organic plant consumption must be considered when using the natural abundance of the stable isotopes of nitrogen as a dietary intake biomarker or a compliance marker. A case-by-case approach accounting for the nitrogen cycle should be used and adapted to the population, community or individuals concerned. We hope that future implications include a cautious context-dependent use of the natural abundance of nitrogen isotopes as a marker of food choices and adherence to nutritional advice.