2.3 Energy content nutritional analysis
We measured energetic value and proximate composition (the proportion of protein content, lipid content, moisture, and percent ash) for sea otter prey items. We used previously established methods to measure energy density using a Parr 6725 semi-micro bomb calorimeter. Standards and replicates were used to confirm consistent calorimeter readings. Lipid content was determined using previously established methods using a sulfo-phospho-vanillin colorimetric analysis. Protein content was estimated by multiplying total nitrogen content by 6.25, which accounts for the nitrogen content of protein . Nitrogen content was measured with a FlashSmart elemental analyzer coupled to a Delta-V continuous-flow isotope ratio mass spectrometer (Thermo Scientific, Waltham, Massachusetts, USA). Carbohydrate content was not assessed as it is assumed to be negligible in marine invertebrates . Ash content was processed at 600°C and measured with a LECO Thermogravimetric Analyzer 701 (TGA) dryer. Only samples with > 1 g of dried material were able to be combusted for percent ash content.