Cholesteryl oleate

Neutral (pH 7) and acidic (pH 4.5) CE hydrolase (CEH) and TG hydrolase (TGH) activities in liver lysates were measured using radioactively labeled substrates as previously described (17 ) with minor modifications. Briefly, half of the largest liver lobe was lysed in lysis buffer supplemented with 1 mM dithiothreitol and a protease inhibitor cocktail. After centrifugation, the supernatant was collected, and protein concentrations were determined as described earlier. To measure CEH activities, the substrate contained 200 μM cholesteryl oleate and 0.04 μCi cholesteryl [1-¹⁴C]-oleate (Amersham Biosciences) per sample, whereas TGH activities were determined with a substrate containing 300 μM triolein and 0.5 μCi [9,10-³H(N)]-triolein (PerkinElmer) per sample. The substrates for the CEH activity assay at pH 4.5 and 7 and for the TGH activity assay at pH 4.5 were emulsified in 455 μM mixed micelles of phosphatidylcholine and phosphatidylinositol (3:1). The substrate for the TGH activity assay at pH 7 was emulsified in 45 μM of above-mentioned micelles. The reconstituted substrates were mixed with 50 μg of liver lysate proteins diluted in citrate buffer for assays at pH 4.5 and in potassium phosphate buffer for assays at pH 7. The rest of the assay was performed as previously described (18 ), and activities were calculated from the release of FA (19 (link)).

The lipid donor nanoparticle was prepared from a lipid mixture described by Maranhão et al. [16 (link)]. In a vial, 40 mg cholesteryl oleate, 20 mg egg phosphatidylcholine, 1 mg triolein and 0.5 mg cholesterol, purchased from Sigma Aldrich, were mixed. Trace amounts of glycerol tri [9,10(n)-³H] oleate and 4-¹⁴C-cholesterol or [1α,2α(n)-³H]-cholesteryl oleate and L-3-phosphatidylcholine,1-stearoyl-2-[1-¹⁴C] arachidonyl (Amersham, Little Chalfont, UK) were added to the initial solution. The lipids were emulsified by prolonged ultrasonic irradiation in aqueous media and a two-step ultracentrifugation of the crude emulsion with density adjustment by addition of KBr to obtain the nanoparticle. The nanoparticle fraction was dialyzed against a 0.9% NaCl solution.