Phospholipid c test

Plasma levels of TG, total cholesterol, PLs, non-esterified fatty acids (NEFA), and glucose were measured using commercial enzyme assay kits (TG E-test, Wako; Cholesterol E-test, Wako; Phospholipid C-test, Wako; NEFA C-test, Wako; Glucose CII-test, Wako; FUJIFILM Wako Pure Chemical Co., Osaka, Japan).

DOPC, DOPS, NBD‐DOPE, Rho‐DOPE, and Cy5‐DOPE were mixed in chloroform into glass microtubes at various molar ratios. The solutions were evaporated under flowing argon gas, resulting in the formation of lipid film. The films were placed in a desiccator in vacuo overnight to completely remove chloroform. The film was hydrated by adding 250 µL of buffer [20 mm CH₃COOH/CH₃COONa (pH 4.5) or 10 mm Tris‐HCl (pH 7.5)] and incubated overnight at 27 °C. The suspension was extruded through a 100‐nm pore polycarbonate membrane using a mini‐extruder (Avanti Polar Lipids). The lipid concentration was measured using the Phospholipid C‐Test (Wako, Osaka, Japan), which quantifies choline produced by phospholipase D activity.

The Au@PL was prepared on the basis of a previous report [11 (link)]. 100 nm AuNPs were coated with octanethiol by mixing 2 mL of AuNP solution, 1 mL of water, 3 mL of ethanol, 3 mL of chloroform, and 10 μL of octanethiol and stirring for 3 hours at room temperature. The solution was left for 30 minutes until separating into two phases. The bottom phase containing octanethiol-modified AuNPs was extracted into the flask, and the solvent was evaporated under vacuum condition for 3 hours. A methanol/chloroform (v/v = 1/2) solution of DMPC/DMPS (molar ratio 9:1) was added to the flask. The solvent was removed by rotary evaporation at 60°C and then kept under a high vacuum for at least 3 hours. After completely removing the solvent, freezing-thawing treatment was performed five times and crude Au@PL was obtained.
The crude Au@PL suspension was centrifuged at 5000 rpm for 5 minutes to isolate Au@PL from the mixture of Au@PLs and PL vesicles. The precipitated Au@PLs were dispersed in distilled water and the final concentration of PC was 0.45 mM measured with an assay kit (Phospholipid C-Test; Wako Pure Chemical) [13 (link)]. The UV-vis spectra of Au@PLs were measured from 400 to 700 nm by UV-vis spectroscopy (UV-1800, Shimadzu, Kyoto, Japan).

Serum lipids were assayed enzymatically using commercial kits (Cholesterol E-test, Triglyceride E-test, Phospholipid C-Test, Wako Pure Chemical Industries, Osaka, Japan). Liver lipids were extracted by the method of Folch et al. [35 (link)], and then the concentrations of cholesterol, phospholipid, and triglyceride were measured enzymatically.

LLC-PK1 and MES 13 were treated with 0, 0.2, 0.5 and 1 mg TC/mL βVLDL for two days. Lipids were extracted as described above and phospholipid (PL) was measured using a phospholipid C-test (Wako Pure Chemical Industries, Ltd).
The intracellular distribution of neutral lipid and FC after two days of treatment with or without βVLDL was examined using Oil Red O staining and filipin staining, respectively according to the manufacturer’s protocol.

Plasma concentrations of triglyceride, cholesterol, phospholipid, FFA, and glucose were measured with commercial assay kits (Triglyceride E test Wako, Cholesterol E test Wako, Phospholipid C test Wako, NEFA C test Wako, and Glucose CII test Wako, Wako Pure Chemical Industries, Ltd., respectively). Plasma concentrations of adiponectin, insulin, and leptin were measured by enzyme-linked immunosorbent assay using a commercial kit (Mouse/Rat Adiponectin ELISA kit, Otsuka Pharmaceutical, Co. Ltd., Tokyo, Japan; Rat Insulin ELISA kit, Morinaga Institute of Biological Science, Kanagawa, Japan; and Mouse/Rat Leptin ELISA kit, Morinaga Institute of Biological Science, respectively). Plasma concentration of NOx was measured by detecting nitrogen dioxide and nitrogen trioxide, stable metabolites of nitric oxide, using a commercial kit (NO₂/NO₃ Assay kit-FX, Dojindo Laboratories, Kumamoto, Japan) after deproteinization of plasma by filtration using an Amicon Ultra Centrifugal Filter (Merck Millipore, Ltd., Carrigtwohill, County Cork, Ireland). Plasma specific activity of angiotensin converting enzyme (ACE) was measured with a commercial kit (ACE color Fujirebio, Inc., Tokyo, Japan). All measurements were performed in accordance with the manufacturer’s instructions. The primary outcomes were adiponectin, NOx, and ACE, and the secondary outcomes were the others.

Serum ALT and AST levels were analyzed, as previously described^{59 ,60} . Serum concentrations of glucose, FFA, total-cholesterol, HDL-cholesterol, triglyceride, total protein, phospholipids, and ALP were measured using commercially available assay kits by FUJIFILM Wako Pure Chemical Corporation (Glucose CII-test Wako, Cholesterol E-test Wako, HDL-Cholesterol E-test Wako, NEFA C-test Wako, Triglyceride E-test Wako, Phospholipid C-test Wako, and LabAssay ALP).