Cobas c111 system

The total cholesterol, HDL and triglyceride contents of the plasma samples were analyzed using an enzymatic colorimetric method (Roche, Basel, Switzerland) on the Cobas C111 system (Roche Diagnostics, Basal, Switzerland).

Casein and BW-WM protein were added Trizma buffer (50 mM, pH 8.0) and hydrolysed using trypsin from bovine pancreas (T1426 from Sigma) at 45 °C for 4 h as recommended by Shalaby et al. [37 (link)]. The two blue whiting protein hydrolysates (BW-HA, BW-HP) were not hydrolysed with trypsin prior to analyses. Protein in hydrolysates were quantified on the Cobas c111 system (Roche Diagnostics GmbH, Mannheim, Germany) using the TP2 kit from Roche. Renin inhibition was measured using the Renin Assay Kit (MAK157, from Sigma-Aldrich) as described in the user manual. ACE-inhibition was measured using the method by Shalaby et al. [37 (link)], as previously described [20 (link)].

Plasma glucose content was determined applying an enzymatic reference method GLUC assay (Roche Diagnostics GmbH, Mannheim, Germany) on a Cobas c111 system (Roche, Germany).

All analytic processes were conducted according to the manufacturer’s instructions. Plasma glucose content (unit: mmol·l^-1) was determined applying an enzymatic reference method (Roche Diagnostics, Switzerland) on a Cobas c111 system. Plasma insulin concentrations (unit: pmol·l^-1) were measured with an enzyme-linked immunosorbent assay (ELISA) (Dako, Glostrup, Denmark) and plasma glucagon concentrations (unit: pg·ml^-1) were determined by a radioimmunoassay (RIA) kit (EMD Millipore Corporation, Billerica, MA, USA). Glycosylated haemoglobin (HbA_1C) was determined applying high-performance liquid chromatography (HPLC) on the TOSOH G8 (Tosoh Bioscience, Tokyo, Japan). Health status markers, fasting total cholesterol (TC), high-density lipoprotein (HDL) and triglyceride (TG) were determined using the immunonephelometry procedure (Siemens, Erlangen, Germany) on the Vista Dimension 1500 system. Low-density lipoprotein (LDL) was estimated by using the Friedewald equation. Except for glucose, all samples were analysed in duplicate.

Lipids were extracted from the liver, skeletal muscle and faeces by the method of Bligh and Dyer,^{18 (link)} evaporated under nitrogen and re-dissolved in isopropanol before analysis on Cobas c111 system (Roche Diagnostics GmbH, Mannheim, Germany) using the appropriate kits from Roche Diagnostics GmbH.

Casein and cod protein meals were added Trizma buffer (50 mM, pH 8.0) and hydrolyzed using trypsin from bovine pancreas (T1426, from Sigma-Aldrich) at 45 °C for 4 h as recommended by Shalaby et al. [42 (link)]. ACE-inhibition was measured using the method by Shalaby et al. [42 (link)], as previously described [43 (link)]. Renin inhibition was measured using the Renin Assay Kit (MAK157, from Sigma-Aldrich) as described in the user manual. Protein in hydrolysates were quantified on the Cobas c111 system (Roche Diagnostics GmbH, Mannheim, Germany) using the TP2 kit from Roche.

Animals were fasted for 6 hours, anesthetized with 2% isoflurane (Schering-Plough, Kent, UK) and blood was collected by heart puncture before the mouse was euthanized. The blood was centrifuged, EDTA-plasma separated and stored at −80 °C prior to further analysis. Liver, epididymal white adipose tissue (eWAT), inguinal WAT (iWAT), skeletal muscle (gastrocnemius) and BAT were collected and immediately frozen in liquid nitrogen and stored at −80 °C until further analysis.
Fasting EDTA-plasma samples at study endpoint were analyzed for Total Cholesterol, HDL Cholesterol, LDL Cholesterol, Triglycerides, Non-esterified fatty acids (NEFA) using the Cobas C111 System (Roche Diagnostics GmbH, Mannheim, Germany). Standard kits were used for all except for NEFAs that were analyzed using the NEFA FS kit (DiaSys, Diagnostic Systems GmbH, Germany). Lipids were measured by enzymatic colorimetric assays with specific reagents from Roche Diagnostics for total cholesterol (CHOL2, Cat. No. 04718917190), triglycerides (TRIGL, Cat. No. 04657594190), HDL cholesterol (HDL, Cat. No. 05401488190), and LDL cholesterol (LDL C, Cat. No. 04657578190). Insulin (Cat. No. 90080) and Leptin (Cat. No. 90030) were measured by ELISA from (Crystal Chem, Downers Grove, IL, USA).