Volunteers of both genders were recruited from primary health care centers in Campinas (SP-Brazil) and Ambulatório de Dislipidemia do Serviço de Endocrinologia e Metabologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. The study was approved by the Research Ethics Committee of UNICAMP School of Medicine under nº 120/2007 and Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo under nº 149/7. All participants were informed of the objectives of the protocol and signed an informed written consent according to research protocols approved by the Ethics Committee of HCFMUSP and UNICAMP.
The study included nonsmokers, asymptomatic individuals with body mass index (BMI) lower than 30 kg/m2, without regular use of any medications that interfere with lipid metabolism and daily intake of alcohol lower than 14 g, as previously described .
Subjects were selected with plasma HDL-C concentrations below 40 mg/dL (Low HDL, 7males and 5 females), above 40 mg/dL and below 60 mg/dL (Control, 4males and 6 females), or above 60 mg/dL (High HDL, 7males and 8 females) corresponding to values below the 10th percentile and above the 90th percentile of the Brazilian population. The subjects’ ages were between 20–74 years and body mass index (BMI) below 30 kg/m2. The exclusion criteria were obesity, diabetes mellitus, metabolic syndrome, thyroid function disorders, liver and kidney failures, smoking, alcohol abuse and use of medications that might interfere with the metabolism of cholesterol.
Blood was drawn after 12 h fasting period into tubes containing ethylenediamine tetraacetic acid (EDTA) (10%). Plasma was immediately separated and added benzamidine 2 mM (5 µL/mL), gentamycin + cloranphenicol 15 mM (20 µL/mL), phenyllmethyl sulfonil fluoride 0,5 mM (0,5 µL/mL) and aprotinin 10 mg/mL (5 µL/mL) and butylated hydroxytoluene (BHT). Plasma lipoproteins very-low-density lipoproteins (VLDL), low-density lipoproteins (LDL), high-density lipoproteins (HDL) and lipid free fraction (LFF) were separated by sequential ultracentrifugation using a Beckman Model L-8 ultracentrifuge 50 Ti rotor (Beckman Instruments, Palo Alto, CA, USA) . Plasma and lipoprotein NCSPCS (desmosterol, lathosterol) and phytosterols (campesterol and sitosterol) were measured in samples (100 µL) added 5α-cholestane (1 µg) as the internal standard, hydrolyzed with KOH in ethanol (1 mol/l, 1 ml) at 60 °C (1 h) and extracted with hexane. Sterols were derivatized with a sylilating solution (pyridine and BSTFA (N,O-bis (trimethylsilyl) trifluoroacetamide) + 1% TMCS (trimethylchlorosilane) (1:1, v/v) (Supelco 33155-U) for 1 h at 60 °C 
Plasma and lipoproteins oxysterols (24-OHC and 27-OHC) were measured according S. Dzeletovic et. al. (modified) . Internal standard (100 ng of 24-hydroxycholesterol-d7 and 27- hydroxycholesterol-d7 in chloroform; Avanti Polar Lipids, Alabaster, USA) was added in samples and oxysterols measurements were performed after alkaline hydrolysis adding a mixture of 10 mL of absolute ethanol and 0.4 M of potassium hydroxide overnight, at room temperature. The pH was adjusted to 7 with phosphoric acid followed by 20 mL of chloroform and 6 mL of water. After vigorous shaking and centrifugation at 4◦C, the aqueous phase was removed and the organic phase evaporated. The lipid extract was dissolved in toluene (1 mL). Oxysterols were separated from cholesterol by solid phase extraction. Briefly, the sample was applied into the column (Sigma-Aldrich Supelclean LC-Si SPE Tubes SUPELCO, Bellefonte, USA) previously conditioned with 8 mL of hexane. Cholesterol were eluted with 1.5% isopropanol in hexane (8 mL), and oxysterols were further eluted with 30% isopropanol in hexane (6 mL). Finally, the solvent was evaporated and samples were derivatized with 100 µL of pyridine and 100 µL of N,O-bis (trimethylsilyl) trifluoroacetamide with trimethylchlorosilane (BSTFA; Sigma- Aldrich, St. Louis, USA), for 1 h at 60◦C. The derivatized sample (1 µL) was injected into a gas chromatograph coupled to a mass spectrometer (Shimadzu GCMS-QP2010, Kyoto, Japan) by automatic injector and analyzed in selected ion monitoring. The separation was performed on a Restek capillary column (100% dimethyl polysiloxane-RxiR − 1 ms. Cat. #13323), 30 m, internal diameter 0.25 mm, for 30 min, using helium as mobile phase, with constant linear velocity of 44.1 cm/sec. The oven started at 240◦C with increment of 5◦C/min, for 7 min up to 290◦C. The mass spectrometer operated in impact electron mode at an ionization voltage of 70 eV with the temperature of the ion source at 300◦C.
The quantification was performed comparing the peak areas of the standard curve and corrected for internal standards . Plasma non-cholesterol sterols (µg) and oxysterols (ng) were expressed as ratio of plasma total cholesterol (mg).
The results were expressed as mean ± SD or the median (variation). Differences between groups were compared by Kruskal Wallis (p < 0.05) and Dunn's multiple comparison with correction by Bonferroni post hoc (p < 0.017) was performed when appropriate. Different letters represent statistically significant in the post-test. Gender distribution were compared by Chi-squared test (p < 0.05)