Computational protocol: 1H-NMR metabolomic study of whole blood from hatchling loggerhead sea turtles (Caretta caretta) exposed to crude oil and/or Corexit

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Protocol publication

[…] Data were pre-processed using ACD Labs 12.0 1D NMR Processor (Advanced Chemistry Development, Toronto, Ontario, Canada). This included zero-filling to 16 384 points, phase correction, baseline correction and alignment of the reference TSP signal. Although formate was added as an additional standard, TSP was used as the reference on the expectation that protein concentrations were relatively low in these samples [], and the TSP peaks were consistent and narrow across spectra. For data analysis, residual water and downfield of 9.0 ppm were removed. Two methods of integration were used and compared; intelligent bucket integration with a bin width of 0.03 ppm and integration of identified peaks only. The common integrals for each sample using intelligent bucket integration were normalized by calculating the sum of the integrals for all bins and then dividing each bin by the sum, such that the integrals for each single sample summed to 1. These integrals were subsequently weighted according to sample volume. The common integrals for each sample using identified peaks only were weighted to the TSP integral for each sample and blood volume. Peak identification was performed using Chenomx NMR Suite 8.1 (Chenomx, Edmonton, Alberta, Canada) and The Human Metabolome Database (HMDB; []). [...] Principal components analysis (PCA) was performed using both intelligent bucket integration and integrals of identified peaks only with JMP Pro12 (SAS Institute, Inc., Cary, NC, USA). For the PCA using intelligent bucket integration, dark regions from −1.5 to 0.5 ppm, 4.7 to 4.9 ppm, and 8.25 to 11 ppm were selected. These dark regions eliminated areas without metabolites, NMR standards and the water peak from analysis. Integrals of identified metabolites were compared among treatments using Kruskal–Wallis tests (Statistix 8.1, Statistix, Inc., Tallahassee, FL). […]

Pipeline specifications

Software tools Chenomx NMR Suite, JMP Pro
Databases HMDB
Applications Miscellaneous, NMR-based metabolomics
Organisms Caretta caretta
Chemicals Lactic Acid, Propylene Glycol, 3-Hydroxybutyric Acid