6890 plus

Fatty acid methyl esters (FAMEs) were analyzed by using a Hewlett-Packard 6890 Plus gas chromatograph equipped with split/splitless injector and flame ionization detector (FID). Separation was performed on a DB-23 capillary column (60 m × 0.25 mm i.d., 0.25 µm film thickness) coated with polar stationary phase (50% cyanopropyl, 50% methyl polysiloxane) (Agilent Technologies, Santa Clara, CA, USA). The GC conditions were as follows: oven temperature, 190 °C isothermal for 20 min; injector temperature, 250 °C; detector temperature, 275 °C; carrier gas, helium; carrier gas flow rate, 1.5 mL/min flow rate; flow split, 85:1; injection volume, 1.0 µL. Data were collected using the HP workstation, and fatty acid methyl esters were identified by comparing retention times with those from reference standard mixtures: lauric, myristic, palmitic, palmitoleic, margaric, heptadecenoic, stearic, oleic, linoleic, linolenic, arachidic, and gadoleic acids. The results were expressed as percentage of the total fatty acid methyl ester content determined in this study (i.e., sum of 12 FAMEs). Method repeatability was assessed by analyzing ten times the same extract, while reproducibility was estimated by analyzing ten extracts from the same subcutaneous tissue sample.

Sterols were measured by gas chromatography equipped with a flame ionization detector (GC-FID) (Hewlett Packard 6890 plus), and with a capillary column (DB-XLB 30 m × 0.25 mm i.d. × 0.25 μm; Agilent Technologies, Amstelveen, Netherlands). Extraction of cholesterol and non-cholesterol sterols was performed based on Mackay et al. [19 (link)]. Briefly, a 100 μL plasma sample was saponified with 1 ml of 90% ethanolic sodium hydroxide for 1 h at 60 °C. 5α-cholestane and epicoprostanol were used as internal standards. After two rounds of cyclohexane extraction, samples were derivatized with 30 μL of TMS reagent (pyridine, hexamethyldisilazane and trimethylchlorosilane (9:3:1, v/v/v)). Samples were injected into GC-FID; cholesterol and non-cholesterol sterol peaks were integrated (OpenLab CDS ChemStation Edition; Agilent Technologies, Santa Clara, CA, USA) and their concentrations were calculated relative to the internal standard 5α-cholestane. Non-cholesterol sterol concentrations were standardized for cholesterol concentrations, as determined within the same GC run and expressed as μmol/mmol cholesterol.

Serum non-cholesterol sterol concentrations, campesterol, sitosterol, cholestanol and lathosterol, were measured by gas chromatography (GC) equipped with a flame ionization detector (GC-FID) (Hewlett Packard 6890 plus), using a capillary column (DB-XLB 30 m × 0.25 mm i.d. × 0.25 um; Agilent Technologies, Amstelveen, Netherlands). Extraction of cholesterol and non-cholesterol sterols was performed as described by Mackay et al. [26 (link)]. Briefly, 100ul serum sample was saponified with 1 mL of 90% ethanolic sodium hydroxide for 1 h at 60 °C. 5α-cholestane and epicoprostanol were used as internal standards. After two rounds of cyclohexane extraction, samples were derivatized with 30ul of TMS reagent [pyridine, hexamethyldisilazane and trimethylchlorosilane (9:3:1, v/v/v)]. Samples were injected into the GC-FID, and cholesterol and non-cholesterol sterol peaks were integrated (OpenLab CDS ChemStation Edition; Agilent Technologies, Santa Clara, USA), and their concentrations were calculated relative to the internal standard 5α-cholestane concentration. Non-cholesterol sterol concentrations were standardized for cholesterol concentrations, as determined during the same GC run and expressed as umol/mmol cholesterol.