Increased concentration of 7-ketocholesterol and 7β-hydroxycholesterol in HDL from young anabolic androgenic steroid users

Background: Oxysterols are cholesterol oxidative derivatives that play an important role in cell cholesterol homeostasis. However, HDL particles enriched with oxysterols, especially the 7-ketocholesterol, have lower ability to mediate cell cholesterol removal by the reverse cholesterol transport. Anabolic androgenic steroids (AAS) misuse is associated with diminished cholesterol eux and increased atherogenesis in young men. Unknown is whether the concentration of oxysterols is altered in AAS users. Methods: study from a retrospectively registered study NCT03450837.


Introduction
Oxysterols are derived from the enzymatic and/or non-enzymatic oxidation of the ring structure and side chain portion of the cholesterol molecule that plays an important role in cell cholesterol homeostasis (1)(2)(3). The 27-hydroxycholesterol (27-OH) is related to cell cholesterol homeostasis by controlling the expression of genes involved in cholesterol biosynthesis and excess cholesterol exportation. Moreover, 27-OH is considered an adding route to the reverse cholesterol transport due to the solubility through the plasma membrane (4).On the other hand, 7-ketocholesterol (7-K), 7β-hydroxycholesterol (7β-OH), and 7αhydroxycholesterol (7α-OH) are the most prevalent oxysterols in atherosclerotic lesions and closely related to in ammation, oxidation, monocyte recruitment and apoptosis in the arterial wall compartment.
Moreover, increased7-K alters cellular plasma membrane leading to an impairment of the cholesterol e ux, which is the rst step of the reverse cholesterol transport (RCT) that drives cholesterol to the liver for excretion into bile and feces (5).
Recent studies show the presence of subclinical coronary arterial disease (11,12) and diminished cholesterol e ux capacity mediated by high-density lipoprotein (HDL) in young AAS users (12). Increased oxysterols may be one of the mechanisms involved in the lower HDL functionality in young AAS users.
However, this hypothesis remains to be tested.
In the present study, we evaluated the oxysterols 7-K, 7α-OH, 7β-OH and 27-OH in HDL particles isolated in a subgroup of current AAS users and nonuser involved in strength training, and sedentary men.

Methods
This is a subanalysis from a previous cross-sectional study (12). We evaluated a subgroup of 18 participants (29 ± 5 year): six AAS users (AASU), six AAS nonusers (AASNU) and six sedentary control men (SC). Both groups (AASU and AASNU) were recreational weightlifters or amateur bodybuilding athletes who were recruited from gymnasiums. The inclusion and exclusion criteria were previously described (12).
After alkaline hydrolysis (10 mL of absolute ethanol and 0.4 M of potassium hydroxide, for 2 h at room temperature, pH 7.0) lipids were extracted with 20 mL of chloroform and 6 mL of 0.9 M NaCl following vigorous agitation and centrifugation (750 × g for 15 min at 4 °C). The organic phase was evaporated and diluted in 1 mL of toluene. Oxysterols and cholesterol were isolated by solid phase extraction (Sigma-Aldrich Supelclean LC-Si SPE Tubes SUPELCO, Bellefonte, USA). The sample was applied to a column previously conditioned in 2 mL of hexane, following a wash with 1 mL of hexane. Cholesterol and oxysterols were sequentially eluted by using 8 mL of 1.5% isopropanol in hexane and 6 mL 30% isopropanol in hexane. The solvent was evaporated, and the lipid extract was derivatized by using pyridine: trimethylsilyltri uoroacetamide (BSTFA; Sigma-Aldrich, St. Louis, USA) (1:1, v/v) at 60 °C for 1 h.
One µL of sample was injected into a gas chromatograph coupled to a mass spectrometer (Shimadzu GCMS-QP2010, Kyoto, Japan). The separation was performed on a Restek capillary column (100% dimethyl polysiloxane-Rxi®-1 ms. Cat. #13323), 30 m, internal diameter 0.25 mm, for 30 min, using helium as mobile phase, with a constant linear velocity of 44.1 cm/sec. The oven started at 240 °C for 7 min with an increment of 5 °C/min, up to 290 °C. The mass spectrometer was operated in single ion monitoring, an ionization voltage of 70 eV with the temperature of the ion source at 300 °C (13,14). The intra assay coe cient of variation was 19%.

Statistical Analysis
Data are presented as mean ± standard deviation (SD). The Shapiro-Wilk test was used to evaluate the normal distribution of the variables. The parametric data were analyzed by one-way ANOVA analysis of variance. When a signi cant difference was found, the Scheffé post-hoc comparison test was used. P < 0.05 indicates statistical signi cance.

Discussion
Oxysterols participate in the regulation of several physiological process including those related to intracellular cholesterol homeostasis (1)(2)(3). On the other hand some species of oxysterols are more related to in ammation, apoptosis and atherosclerotic plaque instability. Increased oxysterols plasma concentrations are observed in individuals with hypercholesterolemia and atherosclerosis and are associated with the degree of coronary stenosis (15). High oxysterols concentrations have been reported in the atherosclerotic plaque. In addition, they are associated with the progression of the lesion (16). The accumulation of 7-K is closely related to the expression of endoplasmic reticulum stress markers in atherosclerotic macrophages as well as to the rupture prone areas in lesion(17). Furthermore, previous studies showed that HDL particles enriched with oxysterols have lower ability to remove cellular cholesterol(4). The intracellular accumulation of 7-K alters plasma membrane leading to the impairment in cholesterol e ux (5). In the present study, we found increased levels of 7β-OH and 7-K in HDL isolated from AAS users. This response may contribute to the lower ability of HDL in removing cell cholesterol and trigger a higher pro-oxidant and pro-in ammatory effect in young male under AAS abuse. In fact, we previously found a diminished cholesterol e ux mediated by HDL and coronary artery disease in young male AAS users (12).
The present study has limitations. This is an exploratory and observational analysis of a subgroup of participants. We included only men and the results should be interpreted with caution in women. However, Page 5/8 the results were highly signi cant and relates to previous data regarding HDL loss of function.

Conclusion
Anabolic androgenic steroids abuse increases the amount of 7β-OH and 7-K in HDL, which may explain, at least in part, the impairment in macrophage cholesterol e ux mediated by HDL and atherosclerotic coronary artery disease previously reported in young AAS users. The clinical implication of this nding should be considered in future, larger, long-term studies.  Figure 1 Concentration of oxysterols, 7-ketocholesterol (7-K), 7β-hydroxycholesterol (7β-OH), 7α-hydroxycholesterol (7α-OH) and 27-hydroxycholesterol (27-OH) in HDL isolated from anabolic androgenic steroids users (AASU), anabolic androgenic steroids nonusers (AASNU) and sedentary controls (SC). The amount of oxysterols was corrected per mg of cholesterol in HDL; *= p <0.05.