Materials and methods for analysis are described in detail in the Ruiz_Cooly et al (2014). Skin tissue samples with enough material (3.5 mg) were selected for compound specific isotope analysis of amino acids (CSIA-AA). Data from 12 samples were obtained for individual amino acid (AA) d15N values, and 9 samples for d13C values. The Southwest Fisheries Science Center/Pacific Islands Fisheries Science Center Institutional Animal Care and Use Committee (IACUC) approved the original animal work that produced the samples. Sex was determined genetically using qPCR sexing assay by the PRD-Genetic Lab at NOAA. These samples consisted of 5 females, 2 males and 2 unidentified individuals possibly corresponding to females or juvenile males. Large adult males were not included.
The relative pattern of AA d15N and d13C values was highly consistent with past work from other organisms and tissues [1-3]. We grouped data as source- or trophic-AAs for d15N values, and essential- or non-essential-AAs for d13C values to increase power in the analysis and evaluate temporal variation. We calculated average values for each AA group. Regression analyses were conducted to evaluate linear relationship between time and each isotopic tracer for both bulk and individual-AA d15N and d13C values.
We hydrolyzed and prepared approximately 3.5 mg of skin as well as a control (Cyanno; bacteria tissue) [1] to quantify d15N values from source- and trophic-AAs and d13C values from essential- and non-essential-AAs. All derivatives were injected with an AA control, N-leucine, to verify accuracy during each run, and analyzed via gas chromatography-IRMS to obtain d15N and d13C values from individual AAs. Each sample was run 3–4 times to maximize accuracy among chromatograms. The associated analytical error among replicates typically <1.0‰ for AAs used in our analysis. For all samples, d15N values were obtained from a total of four source-AAs (phenylalanine, glycine, lysine, tyrosine), and five trophic-AAs (alanine, glutamate + glutamine, isoleucine, leucine, proline). For d13C values, the essential-AAs that we consistently determined were phenylalanine, valine and leucine, and the non-essential-AA were alanine, proline, aspartate + aspartamine, glutamate + glutamine, and tyrosine. Additional AAs were measured for both nitrogen and carbon, but either had chromatographic issues (lost values among samples, high variability) or are known to have unusual and variable isotopic values relatives to others AAs in their category (e.g., threonine).
Cited References:
1. McCarthy, et al, (2007)
2. Popp, et al. (2007
3. Sherwood, et al (2011)
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