Steller sea lion pup plasma metabolites

Steller sea lion pups were captured live on their natal rookeries. They were restrained physically or chemically. Blood samples were drawn from the vein of the caudal gluteal plexus into blood tubes with anticoagulants (ethylenediaminetetraacetic acid or sodium heparin). During field physical examinations, morphometrics (mass, standard length, axillary girth) and sex were recorded for most pups, as well as any observed external abnormalities. Blood samples were kept chilled in the field until centrifuging (3000–3500 rpm for ten minutes), typically within four hours of collection. Plasma aliquots were stored at -20°C during the remainder of the field research trips (3–10 days) and at -80°C thereafter (months to years, maximal interval of 8 years) following return to the laboratory. Plasma-derived [BUN] and [β-HBA] were measured via spectrophotometer (SpectraMax 340PC384, Molecular Devices, San Jose, CA) using commercially-available endpoint assay kits: [BUN] via StanBio Kit #2050 and Sigma Aldrich Kit #66-20 and #MAK006; [β-HBA] via StanBio Kit #2440 and Sigma Aldrich Kits #310 and #MAK001 (StanBio, EKF Diagnostics USA, Boerne, TX; Sigma Aldrich, now Millipore Sigma, St. Louis, MO). Plasma samples with moderate to severe hemolysis were not included. Technical replicates were ≤10% coefficient of variation. Calculations of metabolite concentrations were made using Softmax Pro (v. 4.8) software or the open-access interface MyAssays.com, applying a four-parameter logistic curve. A point-of-care ketometer (Precision Xtra™, Abbott Laboratories, Abbott Park, IL, ketometer precision=0.1 mmol/L [β-HBA]) was also used. Samples measuring between 0.2–0.4 mmol/L via ketometer were further analyzed via the biochemical assay to improve precision around that threshold important to fasting category assignment (threshold=0.3mmol/L; assay precision≈0.01 mmol/L [β-HBA]). Means of the technical replicates are reported in mmol/L for each metabolite.