Free cholesterol fs

Lipids from plasma were measured enzymatically on a Hitachi 917 system (Roche Diagnostics GmbH, Mannheim, Germany) using the cholesterol (Cholesterol CHOD-PAP, 11491458-216), and triacylglycerol (Triglycerides GPO-PAP, 11730711) kit from Roche Diagnostics, and the free cholesterol (Free Cholesterol FS, Ref 113609910930), non-esterified fatty acid (NEFA FS, Ref 157819910935) and phospholipid kit (Phospholipids FS, Ref 157419910930) from DiaSys (Diagnostic Systems GmbH, Holzheim, Germany). Plasma bile acid was measured enzymatically on a Roche Modular P chemistry analyzer (Roche Diagnostica), using the BA kit (Total Bile Acid Assy Kit, 05471605001) from Diazyme (Diazyme Laboratories, Gregg, CA, USA). The fatty acid composition was determined by GC/MS as previously described [47 (link)]. Glucose was measured on Hithachi 917 using the Glucose/HK kit (Roche Diagnostics, Ref 11876899-216). Fasting insulin was measured in two parallels of 10 μL plasma from each rat using a rat/mouse insulin 96 well plate assay ELISA kit (EZRMI-13K) from EMD Millipore (Billerica, MA, USA), according to the manufacturer’s instructions.

At baseline of the lipid kinetic study, two mL of jugular vein blood samples were collected from 24-h fasted dogs into EDTA tubes (Venoject, Paris, France) and then immediately centrifuged (4°C, 2724 g, 10 min) and stored at –80°C for further analysis. Lipoproteins were separated (18) by fast-protein liquid chromatography (GE Healthcare, Pittsburgh, PA, USA). As reported by Kieft et al. [23 (link)], the precision of this method was determined, using human plasma, by calculating both intra- and interassay precision which is respectively 1.4% and 2.2%. TG, TC, unesterified cholesterol (UC), and unesterified fatty acids (NEFA) were assessed using enzymatic methods (cholesterol RTU and triglycerides enzymatique TG PAP150, Biomerieux, Marcy-l’Etoile, France; free cholesterol FS, Diasys, holzheim, Germany; NEFA C, WAKO, Oxoid, Dardilly, France). Cholesteryl ester (CE) concentration was calculated as TC concentration—UC concentration.

Liver and heart lipids were extracted from frozen samples according to Bligh and Dyer [29 (link)], evaporated under nitrogen, and redissolved in isopropanol before analysis. Lipids from liver, heart and plasma were measured enzymatically on a Hitachi 917 system (Roche Diagnostics GmbH, Mannheim, Germany) using the cholesterol (Cholesterol CHOD-PAP, 11,491,458–216), and triacylglycerol (Triglycerides GPO-PAP, 11,730,711) kit from Roche Diagnostics, and the free cholesterol (Free Cholesterol FS, Ref 113,609,910,930), non-esterified fatty acid (NEFA FS, Ref 157,819,910,935) and phospholipid kit (Phospholipids FS, Ref 157,419,910,930) from DiaSys (Diagnostic Systems GmbH, Holzheim, Germany). L-carnitine, trimethyl-lysine, γ-butyrobetaine, palmitoylcarnitine and acetylcarnitine were analysed in plasma samples, some pooled from 2 to 3 animals (n = 3–6), by HPLC-MS/MS as described by Vernez et al. [30 (link)] with some modifications [31 (link)].