DOI: 10.5061/dryad.18931zd4x
title: Lipid extraction alters amino acid composition and bulk, but not amino acid, carbon and nitrogen isotope values

description: Rationale: Concerns exist over observed shifts in value and variance of
 nitrogen isotopes following physicochemical extraction of lipids from
 organic matter. The mechanisms behind these apparent changes in bulk
 tissue δ15N values are not fully understood yet have major implications
 for analytical costs and integrity of data interpretations. Methods:
 Changes in proximate analysis, amino acid composition, C:N ratios, bulk
 tissue and amino acid δ13C and δ15N values, and resulting isotope‐based
 food web metrics were compared between lipid‐intact and lipid‐extracted
 muscle tissue of fishes spanning &lt;1% to &gt;20% muscle fat
 content to identify mechanisms of nitrogen isotope fractionation
 associated with physicochemical lipid extraction. Results: Bulk δ13C and
 δ15N values increased and %N, C:N ratios and crude protein content
 decreased following lipid extraction. Resulting bulk isotope niche spacing
 and overlap varied significantly between lipid‐intact and lipid‐extracted
 tissues. While amino acid composition significantly changed during lipid
 extraction, particularly for lipid‐associated amino acids (e.g., Glu, Lys,
 Ser), individual amino acid δ13C and δ15N values, and their associated
 compound‐specific isotope analysis of amino acids (CSIA‐AA)‐based food web
 metrics, did not. Conclusions: Physicochemical lipid extraction caused
 significant tissue composition changes (e.g., leaching of amino acids and
 15N‐deplete nitrogenous waste) that affected δ13C and δ15N values and
 tissue %C and %N beyond simply removing lipids. However, lipid extraction
 did not alter individual amino acid δ13C or δ15N values or their
 associated CSIA‐AA‐based food web metrics.
description: Four species of marine fishes with varying levels of muscle total
 lipid content (Table 1) were purchased from Rhode Island fish markets:
 wild-caught Atlantic cod (<em>Gadus morhua</em>, hereafter
 “cod”) from southern George’s Bank, wild-caught bluefish
 (<em>Pomatomus saltatrix</em>, hereafter “bluefish”) from
 southern George’s Bank, wild-caught Atlantic herring (<em>Clupea
 harengus</em>, hereafter “herring”) from “the Northwest Atlantic”,
 and Norwegian farmed “organic” Atlantic salmon (<em>Salmo
 salar</em>, hereafter “salmon”). Dorsal muscle tissue was isolated
 from each fish fillet, frozen at -20 °C, and then lyophilized for 72 hrs
 prior to homogenization with mortar and pestle. Freeze-dried, homogenized
 samples were subdivided into two aliquots, one with original lipid content
 intact and one with lipids extracted. For the latter, lipids were removed
 using a modified version of the classic Bligh and Dyer method. Briefly,
 freeze-dried, homogenized samples were soaked for 30 min at room
 temperature in a chloroform:methanol solution (2:1v/v ratio) with solvent
 volume 5x sample volume, vortexed for 30 s, left undisturbed to settle for
 30 min, and centrifuged for 10 min at 3400 rpm. The supernatant containing
 solvents and lipids was removed and the process was repeated (at least 3
 full cycles) until the supernatant was clear and colorless following
 centrifugation. Samples were re-dried at 60 °C for 24 h to remove any
 remaining solvent. Proximate analysis of crude protein,
 crude fat, crude fiber, crude carbohydrate, ash, and moisture and amino
 acid compositions (16 individual amino acids) were conducted on both lipid
 intact and lipid extracted muscle samples from all four species (n = 5
 replicates per treatment per species) at the New Jersey Feed Laboratory,
 Trenton, New Jersey, USA. For bulk stable isotope
 analysis, 1.0 ± 0.1 mg of each sample was analyzed for bulk
 δ<sup>13</sup>C and δ<sup>15</sup>N values using a
 PDZ Europa ANCA-GSL elemental analyzer interfaced to a PDZ Europa 20-20
 isotope ratio mass spectrometer (IRMS; Sercon Ltd., Cheshire, UK) at the
 UC Davis Stable Isotope Facility, Davis, CA, USA. Data are reported with
 standard d notation in per mil (‰) units relative to Vienna PeeDee
 Belemnite and Air for carbon and nitrogen, respectively. Long term
 standard deviation is ± 0.2‰ for d<sup>13</sup>C and ± 0.3‰
 for d<sup>15</sup>N analyses. For CSIA-AA, 2.0 ± 0.1 mg of
 each sample and a long-term laboratory working standard of Atlantic cod
 muscle were acid hydrolyzed to isolate free amino acids in 6N HCl at 110°C
 for 20 hrs in tubes under N<sub>2</sub> headspace to prevent
 oxidation during hydrolysis. Acid hydrolyzed samples, the working
 laboratory standard, and a laboratory amino acid reference standard
 containing all target amino acids with known isotopic values
 (Sigma-Aldrich) were spiked with an internal Nor-Leucine standard and then
 derivatized to N-trifluoroacetic acid isopropyl esters prior to CSIA-AA
 according to established protocols in McMahon et al. (2018).
 Derivatized samples and standards were injected (240°C) in split
 mode (15:1 split, d<sup>13</sup>C analyses) or splitless mode
 (d<sup>15</sup>N analyses) onto a BPX5 column (60m length,
 0.32mm ID, 1µm film thickness) for separation in a Thermo Trace 1310 GC,
 combusted into CO<sub>2</sub> or N<sub>2</sub> gas
 via a GC-IsoLink II oxidation/reduction furnace (1000°C), passed through a
 liquid nitrogen cold trap (d<sup>15</sup>N analyses only), and
 analyzed with a Thermo Scientific Finnigan Delta V Plus IRMS at the
 University of Rhode Island - Graduate School of Oceanography.
 Standardization of runs was achieved using intermittent pulses of
 a CO<sub>2</sub> or N<sub>2</sub> reference gas of
 known isotopic composition. All CSIA-AA samples were analyzed in
 triplicate bracketed by the laboratory amino acid reference standard and
 the laboratory working standard to correct for the introduction of
 exogenous carbon (δ<sup>13</sup>C only) and kinetic
 fractionation associated with derivatization (δ<sup>13</sup>C
 and δ<sup>15</sup>N) following Yarnes & Herszage
 (2017). The long-term reproducibility of the laboratory working standard,
 δ<sup>13</sup>C ± 0.4‰ and δ<sup>15</sup>N ± 0.5‰,
 (calculated as the long-term SD across &gt;100 separate full analyses,
 averaged across all individual AAs), provides an estimate of full protocol
 reproducibility (hydrolysis, derivatization, and GC-C-IRMS analysis). For
 δ<sup>13</sup>C analyses, 13 individual amino acids were
 measured and classified as essential (minimal trophic discrimination):
 isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met),
 phenylalanine (Phe), threonine (Thr), valine (Val) or non-essential
 (variable trophic discrimination): alanine (Ala), aspartic acid +
 asparagine (Asx), glutamic acid + glutamine (Glx), glycine (Gly), proline
 (Pro), serine (Ser). For δ<sup>15</sup>N analyses, 14
 individual amino acids were measured and classified as source (minimal
 trophic discrimination): arginine (Arg), Lys, Met, Phe; trophic (large
 trophic discrimination): Ala, Asx, Glx, Ile, Leu, Pro, Val; and metabolic
 (variable trophic discrimination): Gly, Ser, Thr.
 Offsets in tissue %C and %N, C:N ratios, proximate analysis
 (protein, fat, fiber, carbohydrate, ash, moisture), amino acid
 composition, bulk d<sup>13</sup>C and
 d<sup>15</sup>N values, and amino acid
 d<sup>13</sup>C and d<sup>15</sup>N values were
 compared between lipid intact and lipid extracted muscle samples of the
 four fish species spanning a large range of tissue fat compositions. Total
 hydrolysable amino acid (THAA) d<sup>13</sup>C and
 d<sup>15</sup>N values, as proxies for the carbon and nitrogen
 isotope bulk protein pool, were calculated as the sum of individual amino
 acid d<sup>13</sup>C or d<sup>15</sup>N values,
 weighted by the relative composition of each individual amino acid (Ala,
 Asx, Glx, Gly, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Val representing 64.6 ±
 4.5% of the total protein pool; Table S1).
 d<sup>13</sup>C<sub>THAA</sub> and
 d<sup>15</sup>N<sub>THAA</sub> values were
 compared to bulk tissue d<sup>13</sup>C and
 d<sup>15</sup>N values, respectively, for lipid intact and
 lipid extracted tissues for each species via linear regression to assess
 patterns of changing protein vs. bulk tissue isotope pools.
 To examine how changes in bulk and amino acid
 d<sup>13</sup>C and d<sup>15</sup>N values of fish
 muscle tissue due to lipid extraction impacted ecological interpretation
 of those data, a series of common isotope food web metrics were calculated
 from the same individual muscle samples with lipids intact and lipids
 extracted: 1) isotopic niche width and overlap using bulk tissue
 d<sup>13</sup>C and d<sup>15</sup>N values, 2)
 amino acid carbon isotope fingerprinting, and 3) trophic
 position.  Changes in isotopic niche widths and overlap
 between lipid intact and lipid extracted samples of each fish species were
 calculated using bulk δ<sup>13</sup>C and
 δ<sup>15</sup>N data and the R package
 <em>SIBER</em>. Isotopic niche, which reflects the isotopic
 variation of an organism’s resource use (e.g., diet, habitat), is often
 used as a proxy for ecological niche width. Standard ellipse area (SEA),
 used as a metric of isotopic niche width, was calculated using both
 frequentist (SEA<sub>C</sub>, sample size corrected) and
 Bayesian approaches (SEA<sub>B</sub>) within
 <em>SIBER</em>. The Bayesian approach yields probability
 distributions of isotopic niche width that reflect SEA uncertainty by
 treating isotopic niche region as a probability density function with
 posterior estimates calculated by iteratively fitting multivariate normal
 distributions to each dataset via Markov chain Monte Carlo simulation. The
 model ran two chains of 20,000 iterations, burning the first 1,000 draws
 and thinning every 10 draws. To test for differences in isotopic niche
 size between lipid intact and lipid extracted bulk
 δ<sup>13</sup>C and δ<sup>15</sup>N data, I
 compared each pair of SEA<sub>B</sub> posterior draws and
 quantified the proportion that were smaller (or larger) in magnitude.
 Differences in SEA<sub>B</sub> were considered significant if
 ≥ 95% of posterior draws were smaller or larger than the other (lipid
 intact vs. lipid extracted for each species). I quantified the extent to
 which lipid intact and lipid extracted isotopic niches overlapped for each
 species using the <em>maxLikOverlap </em>function within
 <em>SIBER</em>. As it was hypothesized that the majority of
 the variation in bulk tissue isotope values following lipid extraction
 would come from changes in d<sup>13</sup>C values owing to
 removal of low <sup>13</sup>C lipids, I also examined the
 impact that lipid extraction had on niche overlap using changes in
 d<sup>15</sup>N alone (i.e., d<sup>13</sup>C
 values held constant at pre-lipid extraction values). This test
 facilitated an assessment of niche overlap impact owing to changes in the
 non-target isotope N. I quantified the relative
 contributions of potential primary producer end members to individual
 fishes (n = 5 individuals per treatment per species) using a fully
 Bayesian mixing model approach within the “Mix” - Stable Isotope Analysis
 in R (MixSIAR, Stock et al. 2018) package using essential amino acid
 δ<sup>13</sup>C values (Thr, Ile, Val, Leu, Phe), with species
 as a fixed factor and treatment as a random factor. I used published water
 column phytoplankton and benthic macroalgae as end members for cod,
 bluefish, and herring. For farmed salmon, I also included terrestrial
 plants as a potential end member given the increased use of plant-based
 aquaculture feed for farmed salmon. A low trophic discrimination factor of
 0.1 ± 0.1‰ was selected to reflect the minimal fractionation that occurs
 between diet and consumer essential amino acids.<sup>43</sup>
 The model was run with an uninformative prior using three chains for
 1,000,000 iterations, with a burn-in period of 500,000 iterations and a
 thinning interval of 500. Model convergence was verified using
 Gelman-Rubin (no variables &gt;1.05) and Geweke (&lt;5% of
 variables outside the 95% confidence interval) criteria. Resulting
 posterior probability distributions were used to estimate the proportional
 contributions of the primary producer end members to fishes as a means of
 testing whether potential alterations to individual amino acid
 d<sup>13</sup>C values altered resulting amino acid carbon
 isotope fingerprinting results. Note, this approach identifies the
 relative contribution of primary producer end members at the base of the
 food web supporting these upper trophic level fishes and does not reflect
 direct consumption of these end members. I compared
 source amino acid d<sup>15</sup>N values, a metric of baseline
 nitrogen isotope dynamics, from lipid intact and lipid extracted muscle
 tissue via linear regressions for all species and source amino acids (Arg,
 Lys, Met, Phe). Similarly, I compared trophic positions of individual
 fishes from lipid intact and lipid extracted muscle tissue via linear
 regressions for all species. Trophic positions of individual fishes were
 calculated using a TP<sub>CSIA-AA</sub> approach that is
 internally indexed to the δ<sup>15</sup>N baseline. Based on
 the results from the amino acid carbon isotope fingerprinting mixing
 models (Table S1), I used a fully non-vascular autotroph 𝛽 value (3.3 ±
 1.8‰)<sup>52</sup> for cod, bluefish, and herring along with a
 marine fish food web TDF<sub>Glx-Phe</sub> (6.6 ± 1.7‰). For
 farmed salmon I used a 𝛽<sub>mix</sub> value (-1.45 ± 2.6‰)
 reflecting 52% non-vascular autotroph 𝛽 (3.3 ± 1.8‰) and 48% vascular
 autotroph 𝛽 (-6.6 ± 3.4‰)<sup> </sup>and a composite
 TDF<sub>Glx-Phe</sub> (7.1 ± 1.1‰) reflecting 52% marine fish
 food web TDF<sub>Glx-Phe</sub> (6.6 ± 1.7‰) and 48%
 terrestrial plant TDF<sub>Glx-Phe</sub> (7.6 ± 0.4‰) based on
 their amino acid carbon isotope fingerprinting mixing model data. Measures
 of error in fish δ<sup>15</sup>N<sub>Glx</sub> and
 δ<sup>15</sup>N<sub>Phe</sub> values from
 triplicate analytical analyses, 𝛽, and TDF<sub>Glx-Phe</sub>
 were propagated through the trophic position calculation using R package
 “Propagate v.1.0-6”.
description: # Lipid extraction alters amino acid composition and bulk, but not amino
 acid, carbon and nitrogen isotope values
 [https://doi.org/10.5061/dryad.18931zd4x](https://doi.org/10.5061/dryad.18931zd4x) ## Description of the data and file structure Individual fish data including: common name, Genus species, treatment (lipid intact, lipid extracted), sample ID, percent carbon, percent nitrogen, C:N ratio, crude protein, crude fat, carbohydrates, fiber, ash, and moisture content, amino acid composition (relative %), bulk d13C values, bulk d15N values, total hydrolysable amino acid (THAA) d13C values, THAA d15N values, individual essential amino acid d13C values (mean and SD from triplicate analyses, N = non-essential amino acids, E = essential amino acids) with lipids intact and with lipids extracted, individual amino acid d15N values (mean and SD from triplicate analyses, T = trophic amino acid, S = source amino acid, M = metabolic amino acid) amino acid carbon isotope fingerprinting model results (relative %), and trophic position.