Results
Posttest Subject Questionnaire
Subjective analysis of the posttest subject questionnaire found that all 6 participants appeared to have exhibited full adherence to the supplement protocol, and were able to finish the required dosing regimen and testing procedures with no side effects. Evaluation of the questionnaires also revealed no noticeable changes in dietary habits or physical activity levels during the trial period.
Serum FT and TT Levels
Compared to baseline, posttests showed FT levels to be significantly higher, indicating a significant main effect for FT (p = 0.092). TT levels increased, but the increases were not enough to be deemed significant (p= 0. 1484) (37).
Table 1: FT Levels Baseline v. Post
Group Baseline FT FT After Free Test™ Supplementation
Mean 9.550 pg/mL 16.983 pg/mL
SD 1.807 5.204
SEM 0.738 2.125
N 6 6
Table 2: TT Levels Baseline v. Post
Group Baseline TT TT After Free Test™ Supplementation
Mean 436.67 ng/dL 649.83 ng/dL
SD 200.60 266.31
SEM 81.90 108.72
N 6 6
Table 3: Individual Values By Subject- TT
Subject
Age
Duration of Usage
Baseline TT TT Post
1 29 yr 21 d 450 ng/dL 998 ng/dL
2 34 yr 21 d 306 ng/dL 425 ng/dL
3 18 yr 61 d 689 ng/dL 983 ng/dL
4 34 yr 29 d 321 ng/dL 516 ng/dL
5 31 yr 28 d 196 ng/dL 458 ng/dL
6 49 yr 30 d 658 ng/dL 519 ng/dL
Table 4: Individual Values By Subject- FT
Subject
Age
Duration of Usage
Baseline TT TT Post
1 29 yr 21 d 11.7 pg/mL 25.3 pg/mL
2 34 yr 21 d 10.4 pg/mL 13.0 pg/mL
3 18 yr 61 d 11.0 pg/mL 17.1 pg/mL
4 34 yr 29 d 9.3 pg/mL 18.3 pg/mL
5 31 yr 28 d 7.5 pg/mL 18.1 pg/mL
6 49 yr 30 d 7.4 pg/mL 10.1 pg/mL
Discussion
In this research, we sought to find the effects of Free Test™ supplementation at 4 capsules per day over varying time periods on TT and FT. No adverse side effects were reported by the subjects, and subjective data gathered from posttest questionnaires found that Free Test™ was well-tolerated over time periods ranging from three to nine weeks.
In reference to changes in TT, while subjects did show a directional increase, but this increase was not significant (20). The significant change over the course of the pilot was in FT, which underwent an increase of 78%. The increases in FT were most likely related to several different mechanisms of action in the product. Free Test™ contains Acetly-L-Carnitine, which has been shown to increase nitric oxide (NO) and cyclic GMP (cGMP) levels via elevating acetylcholine levels. NO is important in that it regulates vascular tone, CNS stimulation, and, most importantly, induces the release of luteinizing hormone releasing hormone (LHRH) and regulates cyclic guanosine monophosphate (cGMP) levels. NO also activates the release of LHRH which reaches the pituitary and activates the release of luteininzing hormone (LH) via the activation of neural NO synthase (NOS) in the pituitary gland (17,18,30,32).
Similarly, the fact that the product increases the action of NO and cGMP are important one, as both entities have a stimulatory action on steroidogenesis via increased LH production. Therefore, high cGMP levels also equate to high levels of LH, and when cGMP levels are elevated, it serves as an intermediate in the signaling cascade that ranges from luteinizing hormone (LH) binding to testosterone production. In numerous pathway studies, increases in cGMP increased phosphorylation of the steroidogenic acute regulatory protein (StAR). Steroidogenic acute regulatory protein (StAR) is a Leydig cell cholesterol transfer protein that provides the building blocks for testosterone synthesis. StAR activation is necessary for the stimulation of steroidogenic enzymes involved in the transfer of cholesterol to testosterone. These results suggest that cGMP contributes to the control of basal steroidogenesis (endogenous testosterone production) in Leydig cells through the PKG-dependent modification of the StAR protein and interaction.with LH. Even more important is the fact that LH, via receptors found on the surface of Leydig cells, controls the production and secretion of testosterone. The subsequent binding of LH with its receptor allows signalling through the cyclic AMP pathway via GTP binding proteins. Signal transduction occurs through the protein kinase A pathway as its principal signal transduction mechanism, and this ultimately allows for the release of testosterone after 30-60 minutes of LH stimulation. This is most likely one of the mechanisms that contributed to increased FT (1-3,15-16,32).
Another contributing factor to increased FT has to do with This occurs primarily through elevation of 3,5 cyclic adenosine monophosphate (cAMP), a second messenger important in hormone signaling, via forskolin. One study (Bristow et al, 1984) showed that forskolin was able to increase cAMP levels 4.82 times more than a placebo. Increased cAMP production is responsible for the activation of protein kinase A, which is an enzyme that has positive effects androgen receptor binding even in the absence of other androgens. Increased cAMP also is a signal for steroidogenesis (testosterone production) in the Leydig cells of the testes, by increasing levels of steroidogenic acute regulatory protein (StAR- as mentioned above). By this process, and the fact that high cAMP levels also equate to high levels of luteinizing hormone (the hormone responsible for mediating endogenous spermatogenesis), significant increases in endogenous testosterone production may occur, along with a resulting increase in anabolism and protein synthesis. A 2005 study in The Journal of Obesity Research found that obese men taking 250 mg of 10% forskolin a day for 12 weeks (roughly the dosage included in the daily dosage of Free Test™) experienced an averaged 33% increase in free testosterone levels, averaged a 10 lbs. fat loss per person and increased lean mass an average of 8 lbs (5-12).
The product also seems to have a strong anti-oxidative effect on the testes, which can allow the testes an optimal environment for testosterone production. The combination of N-Acetyl-Cysteine and selenium has been shown in recent studies to cause elevations in FT. Glutathione is a tripeptide thiol found in all cells of the body, and is responsible for regulating protein synthesis and detoxifying cell structures. Selenium is needed for the detoxifying enzyme glutathione peroxidase, and NAC significantly increases glutathione levels. Increases in glutathione and glutathione peroxidase seem to be negatively correlated with cytokine release- as levels of these anti-oxidants increase, cytokine levels decrease, and several types of cytokines have been linked to testicular impairment via decreasing LH levels. This effect is readily apparent in the male reproductive system, as several studies have shown that NAC can reverse testicular damage cross-indicated with cytokine release, and cytokine-related testicular suppression. This alone can allow for conditions that are extremely favorable for optimal testosterone production (13,25-26,33).
The increase in FT levels may also be linked to the inclusion of a natural aromatase inhibitor in the product. 3-Desoxy 7-Keto DHEA has demonstrated strong ability to lower estrogen, as It has shown a high binding affinity (Ki value = 0.22 mM) to the aromatase enzyme, and binds in an irreversible manner. This inhibition allows for the production of less estradiol (E2) and estrone (E1) and allows the user of the compound to maintain a higher level of testosterone- a balance referred to as the Testosterone:Estrogen (T:E) ratio. 3,7-Keto DHEA is unique from other commonly available aromatase inhibitors on the sports supplement market, in that it is a natural metabolite of 7-Keto DHEA, and cannot directly bind to the androgen receptor (AR). 3,7-Keto DHEA (like 7-Keto DHEA) also cannot convert to testosterone, estrogen, or progesterone via any type of enzymatic reaction, so it cannot be considered a prohormone.
Another recent problem with commonly available aromatase inhibitors on the supplement market is the direct conversion of the compound to a controlled substance in the body or during synthesis, either in trace amounts, or full-scale conversion. This does not occur with 3,7-Keto DHEA, as it is formed naturally under various conditions in humans from 7-Keto DHEA and can be readily found in humans in the amount of 5-7 ug/day. The mechanism through which aromatase inhibitors raise testosterone is fairly simple- the HPTA senses low levels of estrogen, and because the body seeks to maintain homeostasis (it likes to maintain at least some estrogen, even in men), there is a concurrent increase in the amount of testosterone that is being produced, as a way to compensate for the low estrogen levels. The increased testosterone levels normally will result in increased estrogen, since there is no estrogen being produced but the brain is essentially tricked into trying to produce more estrogen, so it releases more LHRH and subsequently LH, leading to even higher testosterone levels (23-24).
Contributions
DT designed the study, drafted the manuscript, supervised coordination and data acquisition, and performed the statistical analysis. DO and DL participated in the data acquisition.
Acknowledgments
We thank LabCorp® for assisting with the assays and blood work.
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