Clarus 680 gas

The SCFA concentrations in serum were determined using the acid alcohol methods previously described [34 (link)]. The serum was mixed with an equal volume of butanol (DUKSAN, Seoul, Korea), and HCl was added. The mixture was separated by centrifugation at 15,000 rpm for 15 min at 4 °C and filtered through a 0.45 μm microporous filter. Short-chain fatty acids in the filtrates were detected by gas chromatography (GC, Clarus 680 GAS, PerkinElmer) using an Elite-FFAP 30 m × 0.25 mm × 0.25 μm capillary column, with helium as the carrier gas at a flow rate of 1 mL/min, as previously described [34 (link)].

Terpenoid peak identity was confirmed by GC-MS on a PerkinElmer (Waltham, MA) Clarus 680 gas chromatograph, equipped with a Clarus SQ 8T single quadrupole mass spectrometer. The GC was equipped with PerkinElmer Elite 5 (5% diphenyl polysiloxane) 30 m, 0.25 mm ID, 0.25 μM film thickness column. Hydrogen was used as a carrier gas at a flow rate of 1.5 mL/min. The injector temperature was set to 230°C, and the oven initially at 60°C with ramp to 240°C at a rate of 3°C/min. Injections were 1 μL with a 1:10 split ratio. The mass spectrometer transfer line and source temperatures were 150°C. The mass spectrometer operated with a 3 min solvent delay, after which the instrument scanned from 50 to 500 amu in 1 sec with a 0.05 interscan delay in electron impact positive mode at 70 eV. The instrument was controlled by Turbomass software Version 6.1.0.1963 (PerkinElmer). Compounds were identified based on comparison of retention times with reference standards and GC-MS confirmation.

The homeostasis model assessment for insulin resistance (HOMA-IR) was determined by applying the following formula: fasting insulin (µIU/mL) × fasting glucose (mM)/22.5. The Glucose Analyzer II (Beckman-Coulter, Palo Alto, CA, USA) and Ultrasensitive insulin ELISA kits (R&D Diagnostics, Minneapolis, MN, USA) were employed to measure the serum glucose and insulin concentrations, respectively. ELISA kits from Enzo Life Sciences (Farmingdale, NY, USA) were employed to assess serum 17β-estradiol levels. Additionally, BMD-related biomarkers in circulation were measured using ELISA kits. These biomarkers included rat osteoprotegerin (OPG; Abcam, Cambridge, UK), receptor activator of nuclear factors-κB ligand (RANKL; Abcam), osteocalcin (Abcam), parathyroid hormone (PTH; Abcam), and bone-specific alkaline phosphatase (BALP; MyBioSource, San Diego, CA, USA).
Serum was collected from portal vein blood and combined with acidic ethanol (0.01 N HCl; Duksan, Republic of Korea). SCFA concentrations in the resulting supernatants were measured using a gas chromatograph (Clarus 680 GAS, PerkinElmer, Waltham, MA, USA) equipped with an Elite-FFAP capillary column (30 m × 0.25 mm × 0.25 μm) [27 (link)]. External standards of 1 mM acetate, propionate, and butyrate (Sigma Co., St. Louis, MO, USA) were used for calibration.