Free cholesterol

Total lipid was extracted from intestinal and hepatic tissues essentially as previously described [20] (link). Briefly, 100 mg of tissue was homogenized in 1 ml of isopropanol with tissue disperser followed by vigorous shaking for 45 minutes and then centrifuged at 13,000 RPM for 10 minutes. Supernatant from each sample was equally divided into 2 parts, one was used for triglyceride measurement and the other was used for cholesterol measurement after adding Triton-×100. Intestinal and hepatic triglycerides were measured using an enzymatic kit (L Type Triglyceride M, Wako diagnostics) according to the manufacturer's instructions. Total cholesterol and Free Cholesterol were measured using an enzymatic kit (Cholesterol E and Free Cholesterol, respectively, Wako diagnostics, United states) according to the manufacturer's instructions. The amount of Cholesterol Esters was calculated as the difference between the amount of Free Cholesterol and total cholesterol. To assess the levels of cholesterol and triglycerides in the blood, serum was first collected and then samples from three different animals from the same group were pooled together for further analysis. Measurement of serum cholesterol and trigycerides as well as lipoprotein fractionation in serum samples was achieved by FPLC methods and performed by the Mouse Metabolic Phenotyping Center University of Cincinnati.

Total HDL was isolated by sequential ultracentrifugation (density range 1.063–1.210 g/ml), using potassium bromide (KBr) gradients. HDL composition was determined by measuring the content of total cholesterol (Roche Diagnostics), TG (Roche Diagnostics), free cholesterol (Wako Chemicals, Richmond, VA, USA) phospholipid (Wako Chemicals), apoA-I (Roche Diagnostics) apoA-II (Kamiya Biomedical Company, Seattle, WA, USA), apoE (Kamiya Biomedical Company) and apoC-III (Kamiya Biomedical Company). Composition data are expressed as a % of HDL total mass. PON-1 activity in serum was measured using phenylacetate as a substrate, as described [19 (link)]. Lp-PLA₂ activity was measured using 2-thio-PAF (Cayman Chemical Company, Ann Arbor, MI, USA) as a substrate, according to the manufacturer’s instructions [20 (link)]. To determine the distribution of Lp-PLA₂ among the lipoprotein fractions, apoB-containing lipoproteins were precipitated from serum using dextran sulfate, as described [21 (link)].

Lipoproteins were isolated from thawed plasma by flotation sequential ultracentrifugation, according to density: VLDL (1.006–1.019 g/mL), LDL (1.019–1.063 g/mL), and HDL (1.063–1.210 g/mL). Their lipid and apolipoprotein composition was determined by measuring the content of cholesterol, triglycerides, apoB, apoA-I (Roche Diagnostics, Basel, Switzerland), phospholipids, free cholesterol (Wako Pure Chemical, Osaka, Japan), apoA-II, apoE, and apoC-III (Kamiya Biomedicals, Seattle, WA, USA) in the autoanalyzer. ApoJ was evaluated using commercial ELISA (Mabtech).