To our knowledge, this is the first study that evaluates blood and salivary parameters in OX users, with confirmation by urine metabolite quantification. Although there are differences between groups and timepoints, values of the dosed components were within the reference limits described in the literature. The main results show that there was no correlation among blood, saliva, and urine in OG. However, higher levels of inflammatory cells and lipids in OG are suggestive of inflammation and dyslipidemia.
Regarding education levels of the subjects, our results differ from previous studies , in which most subjects had completed high school, followed by higher education in progress. Our study showed that some of the subjects had completed higher education. However, the current research is in line with other studies, which revealed a higher prevalence of use among people with complete secondary and higher education, as well as those who are specialists in the profession, with aesthetic aspects as the primary motivation for use of AAS [99, 100]. Use of AAS does not depend on educational level; that is, having more or less education is not a determining factor for the use of AAS.
According to the WHO , low levels of HDL cholesterol, as well as high levels of triglycerides and LDL cholesterol, are important risk factors for cardiovascular disease. In fact, improving the lipid profile of individuals is the main preventive measure for cardiovascular diseases. . In the present study, it was found that OX users had reduced levels of HDL cholesterol shortly after use of OX, returning to normal after three months. Despite HDL returning to normal, users had isolated hypertriglyceridemia three months after using OX. These data point to dyslipidemia in OX users.
Interestingly, in a previous study, OX was tested as a cholesterol-lowering drug. The authors proposed 7.5 mg/day for three months, followed by washout for two months, and showed that HDL decreased significantly in patients under these conditions. At the same time, they observed high cholesterol levels . Another study focusing on subjects with metabolic syndrome used oral OX at doses of 10 mg/day for one week, and also found reductions in HDL levels, with marked increases in hepatic ketogenesis. This was due to increased influx of fatty acids into the liver. However, it is not possible to exclude the possibility that short-term administration of OX acts directly on the liver to stimulate the oxidation of fatty acids . In a study aimed at treatment of Klinefelter syndrome with oral OX 0.06 mg/kg/day or placebo for two years, HDL was lowered . In our study, the reduction in HDL was observed, but it was also observed that, immediately after cessation of OX use, HDL levels return to normal, despite differences between the research subjects. Meanwhile, triglyceride levels remained high, pointing to the action of OX on fat metabolism and negative impacts on the cardiovascular system.
Although anabolic steroids in high doses are used for short periods of time , many athletes abuse these anabolic steroids and self-administer doses up to 100- and even 1000-fold more than safe doses, producing circulating testosterone levels two to three orders of magnitude above the healthy male reference range, often for prolonged periods. The maximal anabolic dose of testosterone is not known, but almost certainly vastly exceeds 600 mg of testosterone a week . Excess doses cause dyslipidemia secondary to drugs increasing total cholesterol, create no changes in triglycerides, and decrease HDL [108, 109]. In the present study, we were unable to determine the exact dose of OX used by the volunteers, but we suggest that they were supraphysiological doses, and that they caused a consistent effect of OX on HDL production.
OX has been used for therapeutic purposes in many different situations [78, 79, 110–112]. A study of HIV-infected men that was not controlled for antiretroviral intake treated with OX daily for 12 weeks and reduced HDL for all doses of OX studied . In an animal model, OX elevated triglycerides, reduced HDL, decreased hepatic triglycerides, and elevated levels of non-esterified fatty acids produced by the liver, possibly leading to increased lipidic synthesis of hepatic triglycerides . Additionally, OX may reduce blood triglycerides by further activation of the triglyceride lipase that results in hydrolysis of peripheral triglycerides. Once the intake of OX ceases, triglycerides increase . For the purpose of comparison, we used in this study the reference values of the Brazilian population for triglycerides; the results indicate a significantly increased, almost borderline, risk for cardiovascular disease after ceasing OX use. According WHO classification, individuals who have triglyceride level higher than 180 mg/dl are risk groups for cardiovascular disease. In addition, the results found here for triglycerides were consistent with other studies; that is, increased lipolysis and liberation of free fatty acid with the use of OX [78, 79, 110–112]. However, three months after ending OX use, this variable was higher than the reference standards.
This contradictory result can perhaps be explained by the management of OX, including different doses, different monitoring times, the practice of resistance exercises, days of use, types of cycle, and other variables. Also, the short time of evaluation after use could be a variable. Washout of three or more months may take these parameters back to normal [78, 115]. Despite this, our research presents relevant methodological criteria that were used in the study: comparison with reference values and the detection of metabolite in urine, among others. Studies show that the AAS cycle (duration use)  can last from 10 to 12 weeks [4, 117–119], close to the timeframe of this study. It is important that we evaluated a single, isolated AAS. Other studies have not reported standardization in blood sample collection times [70, 120], in contrast to a previous study that reported collection between 10 and 12 a.m. .
Limitations of our study have to be considered. The results found in our study were different from the studies mentioned above, perhaps because they did not follow the subjects several months after ending the use of AAS. We did not make a standardized dose, or limit cycles, because OX is a controlled drug, and its prescription is provided only by physicians. Another limitation is that the subjects’ family history of atherosclerotic disease was unknown. Micronutrient and macronutrient intake, training intensity, and endurance also were not analyzed. The selection of subjects and randomness of groups would be necessary for subsequent studies.
This study showed, for the first time, reference values of saliva taken from previous research, for comparative purposes about what OX can change, as well as what implications this can have. Additionally, even though there is no consensus in the literature on these values, the use of salivary markers is a non-invasive method, cheaper than blood tests, and may facilitate future studies.