9 10 3h n triolein

Hamster and human CETPs in plasma were quantified as previously described (9 (link)). Briefly, hamster plasma was reacted with TP2 anti-CETP antibody (Ottawa Heart Institute, Ottawa, ON, Canada) and immune complexes were captured on M-280 sheep anti-mouse IgG magnetic Dynabeads (Invitrogen, Carlsbad, CA). Human and hamster CETPs were separated on 8% SDS-PAGE gels (Invitrogen) and transferred to PVDF. CETPs in samples and standards were detected with TP2 antibody followed by a goat anti-mouse IgG HRP secondary antibody (11 (link)). Bands were visualized by Western Lightning Plus ECL reagent (Perkin-Elmer Life Sciences). Chemiluminescence was captured on a digital imager (GE Healthcare, Marlborough, MA) and quantified by ImageJ (https://imagej.nih.gov/ij/). CETP levels are expressed relative to that contained in Ad-null hamsters.
The CETP activity in hamster plasma was quantified as previously described (10 (link)). This assay measured the capacity of plasma samples to promote the transfer of radiolabeled TG and CE from LDL to HDL under conditions where the effect of endogenous lipoproteins on CETP activity is minimized. For these assays, LDL was doubly labeled with [9,10-³H(N)]-triolein and cholesteryl-[1-¹⁴C] oleate (PerkinElmer, Inc.) by a dispersion method (12 (link)).

Dietary lipid absorption of TG and cholesterol was performed as previously described [22 (link)]. Briefly, mice fed chow diet or HF/HCD for 5 weeks were fasted for 4 h, i.p. injected with 500 mg tyloxapol/kg body weight (Merck KGaA; Darmstadt, Germany), and gavaged with 200 μl corn oil containing 2 μCi [9,10-³H(N)]-triolein (Perkin Elmer, Waltham, MA) and 0.5 μCi [1-¹⁴C]-cholesterol (Perkin Elmer, Waltham, MA). Blood was collected 150 and 300 min post-gavage and mice were sacrificed 5 h after administration of the oil bolus. Stomach, duodenum, jejunum, ileum, liver, and feces were collected, lyophilized for 24 h, digested in 1 ml of 1 M NaOH, and radioactivity was measured by liquid scintillation counting.

Tissues were lysed in lysis buffer (100 mM potassium phosphate, 250 mM sucrose, 1 mM EDTA, 1 mM DTT, pH 7), sonicated on ice (3x10 s with 1 min interval), and centrifuged at 1,000 x g and 4°C for 10 min. The supernatant was again centrifuged at 20,000 x g for 30 min at 4°C and the protein concentration was estimated in the lipid-free infranatant (BioRad Laboratories, Hercules, CA). Fifty micrograms of protein were diluted to a final volume of 100 μl with lysis buffer and used to determine neutral TG hydrolase activity. To measure neutral TG hydrolase activity, the samples were mixed with 100 μL of TG substrate [per sample: 0.3 mM triolein, 0.5 μCi [9,10-³H(N)]-triolein (Perkin Elmer, Waltham, MA), 3.5 μg mixed micelles of phosphatidylcholine and phosphatidylinositol (3:1, w:w)]. Each substrate contained NEFA-free BSA at a final concentration of 2% in 100 mM phosphate buffer. After incubation in a water bath for 1 h at 37°C, the reaction was stopped by the addition of 3.25 mL stop solution (methanol:chloroform:n-heptane, 10:9:7, v:v:v) and 1 mL of 0.1 M potassium carbonate (pH 10.5, adjusted with boric acid). The tubes were vortexed for 10-15 s and centrifuged at 800 x g for 15 min at 4°C. The radioactivity in 1 mL of the upper phase was determined by liquid scintillation counting and the release of FAs was calculated.

If not stated otherwise, chemicals were obtained from Merck (Darmstadt, Germany) or Carl Roth GmbH (Karlsruhe, Germany); columns for protein purification were obtained from Cytiva (formerly GE Healthcare Life Sciences (Uppsala, Sweden)). [9,10-³H(N)]-triolein was obtained from PerkinElmer Life Sciences (Waltham, MA, USA). Pierce™ Unstained Protein MW Marker from Thermo Fisher Scientific™ was used as size marker for SDS-PAGE gels. Blue Prestained protein standard broad range marker from New England BioLabs was used for SDS-PAGE and for immuno blotting. Disruption of cells was carried out using a homogenizer (SONOPLUS ultrasonic homogenizer HD 2070, Bandelin, Berlin, Germany).

After a 12 h fasting period, mice were gavaged with 200 μl olive oil containing 0.5 μCi [1-¹⁴C]-triolein (Perkin Elmer, Waltham, MA). Blood was taken 2, 4, and 6 h post-gavage. Six hours after the first bolus, mice were gavaged with 200 μl olive oil containing 2 μCi [9,10-³H(N)]-triolein (Perkin Elmer) and blood was taken 2 and 4 h later. Four hours after the second bolus, mice were sacrificed and tissues were collected, lyophilized, and digested in 1 mL of 1 M NaOH. Radioactivity was measured by liquid scintillation counting.
For determination of the lipid distribution into different lipid classes, lipids were extracted from 20 mg of lyophilized tissue using chloroform:methanol (2:1). Lipid extracts were separated using n-hexane:diethylether:acetic acid (70:30:1, v:v:v), corresponding bands for PL, FC, FFA, TG, and CE were cut, and radioactivity was determined by liquid scintillation counting.

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)).