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Free cholesterol samples were extracted from RBCs through the following procedures; methanol was added to RBCs and samples were heated at 55oC in a shaking water bath for 15 min, before addition of hexane: chloroform (4:1, by volume) and double-distilled water. Thereafter, samples were centrifuged for 15 min at 1500 rpm at 4oC. Supernatants of the samples were dried down under nitrogen and re-dissolved with hexanes and subsequently transferred in injection vials. Samples were injected into an Agilent 6890N gas chromatograph (GC). The GC was connected to a Delta V Plus isotope ratio mass spectrometer (IRMS) through a pyrolysis furnace (alumina tubing, reactor temperature at 1450oC). 

A 30 m capillary column (SAC-5; Supelco, Bellefonte, CA) was installed in the GC which was programmed with the starting temperature at 60oC and isothermal for 1 min and increasing by 25oC/min to 275oC; increasing by 4oC/min to 290oC and isothermal for 3 min; increasing by 25oC/min to 308oC and isothermal for 3 min. Under these conditions, the organic hydrogen from free cholesterol was converted to H2 gas. The enrichment of this H2 gas was then detected by the Delta V Plus IRMS system. Isotopic ratios were expressed as d 2H/1H in per mil against V-SMOW (Vienna Standard Mean Ocean Water). The deuterium enrichment of plasma water was analyzed by temperature conversion elemental analyzer (TC/EA)-IRMS. After correction for free cholesterol pool, FSR was taken as the indicator of the fraction of the cholesterol pool that is synthesized over days 42 and 43 was calculated as:

FSR (%) = δ Deuterium / (δ Plasma water * 0.478) *100 (3)

Cholesterol absorption analysis

The enrichment of 13C-cholesterol detected from free cholesterol extracted from RBCs was used to determine absorption (ABS) using gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS).

The free cholesterol was extracted from RBCs by the procedure as mentioned above. Samples were injected into an Agilent 6890 N gas chromatograph (GC). The GC was connected to a Delta V Plus isotope ratio mass spectrometer (IRMS) through a combustion furnace (alumina tubing, reactor temperature at 940oC). A 30-m capillary column (SAC-5; Supelco, Bellefonte, CA) was installed in the GC which was programmed with the starting temperature at 60oC and isothermal for 1 min and increasing by 25oC/min to 275oC; increasing by 4oC/min to 290oC and isothermal for 3 min; increasing by 25oC/min to 308oC and isothermal for 3 min. Under these conditions, the small portion of free cholesterol was converted to CO2. The 13C content of CO2 was measured by the Delta V Plus IRMS system. Enrichments were then expressed as δ 13C/12C in per mil relative to PeeDee Belemnite (PDB) limestone. The absorption is then calculated with the area under the curve from the graph plotted with δ 13C/12C per mil relative to (PDB) over time.

Body composition analysis

The General Electric Prodigy Body Composition software program, EnCore 2005, was used to analyze scans and generate body composition data.

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