Abstract
Lipid extraction of biomass prior to stable isotope analysis is known to cause variable changes in the stable nitrogen isotopic composition (δ15N) of residual biomass. However, the underlying factors causing these changes are not yet clear. Here we address this issue by comparing the δ15N of bulk and residual biomass of several marine animal tissues (fish, crab, cockle, oyster, and polychaete), as well as the δ15N of the extracted lipids. As observed previously, lipid extraction led to a variable offset in δ15N of biomass (differences ranging from -2.3 to +1.8 ‰). Importantly, the total lipid extract (TLE) was highly depleted in 15N compared to bulk biomass, and also highly variable (differences ranging from -14 to +0.7 ‰). The TLE consisted mainly of phosphatidylcholines, a group of lipids with one nitrogen atom in the headgroup. To elucidate the cause for the 15N-depletion in the TLE, the δ15N of amino acids was determined, including serine because it is one of the main sources of nitrogen to N-containing lipids. Serine δ15N values differed by -7 to +2 ‰ from bulk biomass δ15N, and correlated well with the 15N depletion in TLEs. On average, serine was less depleted (-3‰) than the TLE (-7 ‰), possibly due to fractionation during biosynthesis of N-containing headgroups, or that other nitrogen-containing compounds, such as urea and choline, or recycled nitrogen contribute to the nitrogen isotopic composition of the TLE. The depletion in 15N of the TLE relative to biomass increased with the trophic level of the organisms.
Highlights
Stable isotopes of carbon and nitrogen (δ13C and δ15N) are routinely used in ecology to study a wide range of subjects such as trophic interactions, energy flow, diet composition, feeding habits, and migration
Some studies hypothesized that co-extraction of lipidbound proteins leads to the removal of some amino acids (e.g. [13]), this would imply that the extracted amino acids had a strongly different nitrogen isotopic composition compared to the remaining amino acids [14]
In this study we investigate the cause of the changes seen in the δ15N following lipid extraction of tissues of several marine animals by determining the δ15N of bulk and residual, lipidfree, biomass as well as of the total lipid extract (TLE)
Summary
Stable isotopes of carbon and nitrogen (δ13C and δ15N) are routinely used in ecology to study a wide range of subjects such as trophic interactions, energy flow, diet composition, feeding habits, and migration (see e.g. [1,2]; and references therein). For application of stable isotopes in trophic ecology, lipids are sometimes removed from bulk biomass prior to stable isotope analysis. This is done because lipids are depleted in 13C compared to proteins and carbohydrates due to fractionation during lipid biosynthesis and because different tissues and organisms have variable lipid contents [10,11]. [13]), this would imply that the extracted amino acids had a strongly different nitrogen isotopic composition compared to the remaining amino acids [14] Another hypothesis is that lipid extraction leads to removal of cellular waste products (e.g. ammonia), which have quite different nitrogen isotopic compositions than that of organic nitrogen [21]. No experimental evidence has been provided to support these hypotheses
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