Abstract
The purpose of this study was to determine the effects of 6-OXO, a purported nutritional aromatase inhibitor, in a dose dependent manner on body composition, serum hormone levels, and clinical safety markers in resistance trained males. Sixteen males were supplemented with either 300 mg or 600 mg of 6-OXO in a double-blind manner for eight weeks. Blood and urine samples were obtained at weeks 0, 1, 3, 8, and 11 (after a 3-week washout period). Blood samples were analyzed for total testosterone (TT), free testosterone (FT), dihydrotestosterone (DHT), estradiol, estriol, estrone, SHBG, leutinizing hormone (LH), follicle stimulating hormone (FSH), growth hormone (GH), cortisol, FT/estradiol (T/E). Blood and urine were also analyzed for clinical chemistry markers. Data were analyzed with two-way MANOVA. For all of the serum hormones, there were no significant differences between groups (p > 0.05). Compared to baseline, free testosterone underwent overall increases of 90% for 300 mg 6-OXO and 84% for 600 mg, respectively (p < 0.05). DHT underwent significant overall increases (p < 0.05) of 192% and 265% with 300 mg and 600 mg, respectively. T/E increased 53% and 67% for 300 mg and 600 mg 6-OXO, respectively. For estrone, 300 mg produced an overall increase of 22%, whereas 600 mg caused a 52% increase (p < 0.05). Body composition did not change with supplementation (p > 0.05) and clinical safety markers were not adversely affected with ingestion of either supplement dose (p > 0.05). While neither of the 6-OXO dosages appears to have any negative effects on clinical chemistry markers, supplementation at a daily dosage of 300 mg and 600 mg for eight weeks did not completely inhibit aromatase activity, yet significantly increased FT, DHT, and T/E.
Background
Athletes have long been looking for a way to gain an edge in competition, which has lead many to turn to anabolic steroids. Anabolic steroids are defined as testosterone (TST) or derivatives of TST that are used for their ability to create a state of nitrogen retention and increase fat-free mass by stimulating protein synthesis and/or by decreasing protein breakdown. It has been previously thought that anabolic steroids did not cause an increase in muscle size and strength, but now more recent studies have shown the effect that supra-physiological levels of TST and TST derivatives can increase muscle size and strength in males [1-7].
Once produced, TST does not circulate freely in the blood. Rather, total testosterone (TT) is almost 100% bound in blood to proteins with 40% bound to albumin, 40% bound to a β-globulin called sex hormone binding globulin (SHBG), and 17% is bound to other proteins. The small fraction of TST that is not bound is considered the free testosterone (FT) and is the bioactive component of the hormone. Once bound to its androgen receptor, TST can also be converted to dihydrotestosterone (DHT) by the enzyme 5-α reductase. Alternatively, TST can be converted into estradiol through aromatization by the action of the enzyme aromatase.
There are pro-hormone nutritional supplements available, such as androstenedione, that are precursors to TST, and designer androstenedione derivatives such as androstenediol that are purported to support TST production. These compounds are alleged to increase TST, or to increase the concentration of compounds that can act like TST. There are data in young men demonstrating that the acute sublingual ingestion of androstenedione and androstenediol increased FT and TT up to 180 min [8] and 240 min [9] after ingestion. However, these are acute studies with a small window of TST elevation and do not relevantly reflect the manner in which these types of supplements are typically utilized. More appropriately, there are studies demonstrating that the daily oral ingestion of these compounds over the course of several weeks, in conjunction with resistance training [10,11] and otherwise [12] to be ineffective at increasing endogenous TST levels.
However, in the continued attempts to find supplements that elevate testosterone levels, some companies are manufacturing compounds that have no apparent androgenic activity, but are targeted at increasing the endogenous levels of TST by blunting aromatization and subsequent estrogen synthesis. Aromatase inhibiting drugs are not new and have been used for years as a method of preventing and treating various types of cancer. The drugs operate by suppressing estrogen levels and subsequently increasing endogenous free testosterone levels [increased free testosterone/estrogen (T/E) ratio] and the effects of various pharmacologic aromatase inhibitors such as anastrozole and exemestane on the T/E in both young and old men are well documented [13-15].
Nutritional supplements designed with the intent of inhibiting aromatase activity are relatively new to the fitness community. Examples of these supplements are 6-OXO and Novedex XT, and are alleged to act similar to such aromatase inhibiting drugs as formestane. We have recently shown that eight weeks of supplementation with the aromatase inhibiting nutritional supplement Novedex XT (hydroxyandrost-4-ene-6,17-dioxo-3-THP ether and 3,17-diketo-androst-1,4,6-triene) was effective at increasing serum testosterone and DHT, while only displaying slight increases in estrogen levels in young, eugonadal men [16]. Additionally, compounds with the same (androst-4-ene-3,6,17-trione) and very similar (androst-5-ene-4,7,17-trione) structure as 6-OXO have been shown to irreversibly bind to the aromatase enzyme thereby causing a decrease in estradiol production [17,18]. Therefore, use of these aromatase-inhibiting compounds seem to decrease aromatization and subsequent estradiol synthesis, which apparently increases both TST and T/E.
In view of our previous work [16], there is still little data available on the effects of the various nutritional aromatase inhibiting supplements. Therefore, the purpose of the study was threefold and was to determine the efficacy of an eight week oral supplementation period with either 300 mg/day or 600 mg/day of 6-OXO on: 1) serum hormone levels, 2) serum and urinary clinical safety markers and systemic hemodynamic effects, and 3) serum hormone, serum and urinary clinical safety markers, and systemic hemodynamic effects after a 3-week washout period following both supplementation protocols.
Methodology
Participants
Sixteen apparently healthy, recreationally-active males with a mean age of 26.6 ± 4.9 years, height of 180.2 ± 6.3 cm, body fat of 14.9 ± 4.8 %, and body weight of 87.3 ± 13.2 kg served as participants in the study. All participants were cleared for participation by passing a mandatory medical screening. Participants with contraindications to exercise as outlined by the American College of Sports Medicine and/or who had consumed any nutritional supplements (excluding multi-vitamins) such creatine monohydrate or various androstenedione derivatives or pharmacologic agents such as anabolic steroids two months prior to the study were not allowed to participate. All eligible subjects signed a university-approved informed consent document. Additionally, all experimental procedures involved in this study conformed to the ethical considerations of the Helsinki Code.
Testing sessions
Testing sessions were performed at week 0 and after weeks 1, 3, 8, and 11 in which blood and urine samples were obtained and where body composition, serum hormones, blood and urinary clinical safety markers, and systemic hemodynamic safety markers were evaluated.
Body composition assessment
Total body mass (kg) was determined on a standard dual beam balance scale (Detecto Bridgeview, IL). Percent body fat, fat mass, and fat-free mass were determined using DEXA (Hologic 4200 W, Waltham, MA). Quality control calibration procedures were performed on a spine phantom (Hologic X-CALIBER Model DPA/QDR-1 anthropometric spine phantom) and a density step calibration phantom prior to each testing session. Total body water and compartment-specific fluid volumes were determined by bioelectric impedance analysis (Xitron Technologies Inc., San Diego, CA).
Blood and urine collection
Venous blood samples were obtained from an antecubital vein into a 10 ml collection tube. Blood samples were allowed to stand at room temperature for 10 min and then centrifuged. The serum was removed and frozen at -80°C for later analysis. Urine samples were obtained in mid-stream into a collection container using a standard collection protocol. Urine samples were frozen at -80°C for later analysis. Blood and urine samples were obtained after a 12-hour fast and standardized to the same time of day for each sample.
Supplementation protocol
Based on the premise that there were no muscle performance measurements to be made in the study, such as muscle strength and power, that could otherwise generate a so-called "placebo effect," the decision was made not to utilize a placebo group. Participants were equally divided, matched by age and body mass, and then randomly assigned in double-blind fashion to an eight-week supplementation protocol consisting of the daily oral ingestion of either 300 mg or 600 mg of 6-OXO [androst-4-ene-3,6,17-trione (ErgoPharm, Champaign, IL)]. For the 300 mg group, (n = 8; total body mass = 79.3 ± 13.2 kg, fat-free mass = 67.1 ± 7.9 kg; body fat = 14.7 ± 5.4 %) 100 mg was ingested in the morning with breakfast and 200 mg was ingested with the evening meal. For the 600 mg group, (n = 8; total body mass = 81.1 ± 13.3 kg, fat-free mass = 69.0 ± 12.1 kg; body fat = 15.0 ± 4.2 %) 300 mg was ingested in the morning with breakfast and 300 mg was ingested with the evening meal. For days where no exercise occurred, the supplements were ingested in the same timely fashion. After the supplementation period, a three-week washout period was required during which neither supplement was ingested. Upon analysis of serum testosterone from the baseline blood samples at week 0, it was confirmed that all participants completing the study were eugonadal [10–30 nmol/L (27–107 ng/ml)][19].
Physical activity, dietary intake records, and supplementation compliance
During both the supplementation and washout periods the participants' physical activity and dietary intake were not supervised; however, it was required that all participants keep detailed physical activity and dietary records and not change their routine dietary habits or level of physical activity throughout the course of the study. As such, participants were required to keep weekly physical activity records and four-day dietary records during weeks 0, 1, 3, 8, and 11 and turn in their physical activity and dietary records during each testing session. Each participant returned all of their dietary and physical activity evaluations at the required time points for a 100% compliance rate. The four-day dietary recalls were evaluated with the Food Processor dietary assessment software program (ESHA Research, Salem, OR) to determine the average daily macronutrient consumption of fat, carbohydrate, and protein. In an effort to ensure compliance to the supplementation protocol, participants were supplied with the appropriate number of capsules to be ingested during the time between testing sessions 1, 3, and 8. Upon reporting to the lab at each of the respective testing sessions, participants returned the empty containers and a capsule count was performed if necessary.
Reported side effects from supplements
At the last four testing sessions, participants reported by questionnaire whether they tolerated the supplement, supplementation protocol, as well as report any medical problems/symptoms they may have encountered throughout the study.
Hemodynamic clinical safety markers
At each testing session, participants assumed a supine position for 15 minutes and had their heart rate (HR), systolic blood pressure (SBP), and diastolic blood pressure (DBP) determined to assess the hemodynamic safety of supplementation with 6-OXO. Heart rate was determined by use of a Polar heart rate monitor (Polar, San Ramon, CA), and blood pressure was assessed with a mercurial sphygmomanometer using standard procedures.
Blood and urinary clinical markers
The serum clinical chemistry variables glucose, total protein, blood urea nitrogen, creatinine, BUN/creatinine ratio, uric acid, AST, ALT, CK, LDH, GGT, albumin, globulin, sodium, chloride, calcium, carbon dioxide, total bilirubin, alkaline phosphatase, triglycerides, cholesterol, HDL, and LDL were determined with a Dade Dimension RXL clinical chemistry analyzer (Dade-Behring, Inc., Newark, DE). The whole blood hematological variables, hemoglobin, hematocrit, red blood cell counts, MCV, MCH, MCHC, RDW, neutrophils, lymphocytes, monocytes, eosinophils, and basophils, were determined with an Abbott Cell Dyne 3500 hematology analyzer (Abbott Laboratories, Chicago, IL). The urinary variables glucose, ketones, blood, protein, nitrite, bilirubin, leukocyctes, specific gravity, pH, urobilinogen were analyzed with a Bayer Clinitek 200 Plus urine analyzer (Bayer Diagnostics, Tarrytown, NY).
Serum hormones
Serum TT, FT, DHT, estradiol, estrone, estriol, SHBG, LH, growth hormone (GH), cortisol (Diagnostics Systems Laboratories, Webster, TX), and FSH (Alpco Diagnostics, Windham, NH) using enzyme-linked immunoabsorbent assays (ELISA) and enzyme-immunoabsorbent assays (EIA) with a Wallac Victor-1420 microplate reader (Perkin-Elmer Life Sciences, Boston, MA), and the assays were performed at a wavelength of either 450 or 405 nm, respectively. The average specificity for all assays was 3.5 pg/ml, and in all cases the intra-assay and inter-assay variances were < 10%. Additionally, the amount of cross-reactivity between androstenedione and FT, TT, and DHT was 0.06%, 0.09%, and 1.9%, respectively.
