Lathosterol

Serum triglyceride (TG) , cholesterol, phospholipid, glucose, and non-esterified fatty acids (NEFA) levels were measured using commercial enzyme assay kits (Wako Pure Chemicals) .
Biomarkers related to cholesterol metabolism, such as lathosterol, campesterol, and β-sitosterol, in serum were assayed by a gas chromatography-flame ionization detector (GC-FID) system (GC-17A; Shimadzu, Tokyo, Japan) equipped with a capillary column (SPB TM -1, 60 m×0.25 mm i.d., 0.25 μm thickness, SUPELCO, Bellefonte, PA, USA) using 5α-cholestane (Sigma Aldrich Japan) as an internal standard. In brief, to 250 μL of each serum sample was added 25 μg of 5α-cholestane. Samples were saponified with ethanolic KOH and then the sterols were extracted. The extracted sterols were converted to trimethylsilyl (TMS) ethers using the TMS derivatization reagent (BSTFA ＋TMCS, 99:1, SUPELCO) and then injected to the GC-FID system. The temperatures of the injection port and detector were set at 300℃, respectively. The column temperature was set at 275℃. Helium (purity＞99.995％) was used as the carrier gas. The sterol species were identified by comparison with the retention times of the internal standard and the reference standards (lathosterol, campesterol, and β-sitosterol from Sigma Aldrich Japan) after TMS derivatization.

Methanol, acetonitrile, formic acid, ethyl acetate, n-hexane, squalene, vitamin D3, and 5-cholestane (internal standard, I.S., for ergosterol, brassicasterol, and cholesterol and its precursors) were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Lanosterol was purchased from Nagara Science Corporation (Gifu, Japan), and 7-dehydrocholesterol, desmosterol, lathosterol, cholesterol, and vitamin D2 were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Additionally, 25-

Reference standards with a purity assay greater than 95% were purchased from Sigma Aldrich (Sydney, Australia), including cholesterol, stigmasterol, stigmastanol, brassicasterol, lathosterol, lanosterol, 5b-cholestan-3aol, β-sitosterol, and campesterol (purity assay 65%). All reference standards were prepared in heptane as 500 mg/L stock solutions. An acid hydrolysis solution of 8 M hydrochloric acid diluted in ethanol was prepared before analysis. A saponification solution of 5 M potassium hydroxide (Sigma Aldrich), with the potassium hydroxide dissolved in water, was made up in ethanol to volume. N-O-bis-(trimethylsilyl) trifluoroacetamide with 1% trimethylchlorosilane was obtained from Grace Davison (Columbia, MD); n-heptane, pyridine, and cyclohexane were obtained from Merck (Melbourne, Australia); and boiling chips were obtained from BDH (Sydney, Australia). Ultrapure, Type 1 water was used throughout the experiments and was obtained using a Millipore water purification system (Element A10; Millipore Sigma, Burlington, MA).

Brassicasterol (≥98% purity), campesterol (≥65% purity), desmosterol (≥84% purity), ergosterol (≥95% purity), lanosterol (≥93% purity), lathosterol (≥99% purity), β-sitosterol 3 (≥85% purity), stigmasterol (≥95% purity), and squalene (≥98% purity) were purchased from Sigma Aldrich (St. Louis MO, USA). Cholesterol (≥97% purity) was obtained from Fagron (Barcelona, Spain). Acetonitrile and methanol, UHPLC grade, were acquired from Panreac (Darmstadt, Germany). Potassium hydroxide (KOH) from Fagron (Barcelona, Spain), 96% ethanol from Panreac (Darmstadt, Germany), pyrogallol (≥99% purity) from Sigma Aldrich (St. Louis MO, USA), and heptane from Panreac (Darmstadt, Germany) were used for sample preparation. Three commercially available parenteral lipid emulsions were analyzed.