Cholesterol esterase

The study materials consisted of oleoresin samples of natural carotenoid complex (~10%wt) extracted from the microalga H. pluvialis (Flotow) strain Steptoe (Nevada, USA) with the help of the SC-CO₂ method. The oil obtained from H. pluvialis was used in the preparation of the oleoresin. Chilean Company Atacama Bio Natural Products Inc. (Iquique, Chile) kindly donated oleoresin samples to us. General details of the H. pluvialis production are offered on the company’s website (https://www.atacamabionatural.com/web/index.php/about-us/our-process). All chemicals used in this study were of analytical grade, except for those used for HPLC and GC analyses (high-performance liquid chromatography and gas chromatographic, respectively), which were of chromatographic grade (methanol, acetonitrile, ethyl acetate, water, and n-hexane). The carotenoids astaxanthin, lutein, canthaxanthin, and β-carotene, chemicals for the analysis of total phenols, cholesterol esterase, and reagents for determining antioxidant capacity were procured from Sigma-Aldrich, Santiago, Chile at ≥98% purity. For fatty acid identification and quantification, a standard fatty acid methyl ester (FAME) mix, C4–C24, by Supelco Analytical (Bellefonte, PA, USA) was used, and tripentadecanoin >99% (Nu-Check Pre, Inc., Elysian, MN, USA) was used as the internal standard.

The cell culture reagents were obtained from Hyclone (Logan, USA) and the fetal bovine serum (FBS) was obtained from Gibco (New York, USA). The taurine, cholesterol, cholesterol esterase, cholesterol oxidase, horseradish peroxidase and sodium taurocholate hydrate were purchased from Sigma (St. Louis, MO, USA). The PowerOpti-ECL Western blotting detection reagent was purchased from GenView (Florida, USA). The primary (anti-CYP7A1, sc-25536; anti-c-jun, sc-1694; anti-p-c-jun, sc-7980-R; and anti–HNF4α, sc-8987) and secondary (anti-rabbit immunoglobulin G, sc-2004) antibodies were acquired from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The primary anti-MEK1/2 antibody (D1A5) was obtained from Cell Signaling Technology (Beverly, MA, USA). The primary anti-β-actin antibody was obtained from GenView (Florida, USA). The BCA Protein Assay Kit, Cell Lysis Buffer and PMSF were purchased from Beyotime (Nanjing, Jiangsu, China). All the other chemicals were purchased from Dingguo Changsheng Biotechnology Co. Ltd (Beijing, China).

Plasma total and lipoprotein cholesterol levels were quantified through an enzymatic method described previously, with some modifications [23 (link)]. Briefly, 10 μL of plasma or 300 µL of FPLC fractions were incubated for 30 min at 37 °C with 1% Triton X-100 in 0.5 M Tris-HCl pH 7.6, 50 mM phenol, 50 mM 4-chlorophenol, 0.37% sodium cholate, 0.04% 4-aminoantipyrine, 0.35 U/mL cholesterol esterase, 1.1 U/mL peroxidase (all reagents from Sigma Aldrich, St. Louis, MO, USA), and 0.1 U/mL cholesterol oxidase (Calbiochem, La Jolla, CA, USA). The absorbance was measured at 500 nm in a plate reader and cholesterol concentrations were calculated using a standard curve.