Applied Nutriceuticals® In-House Pilot Work, April 2010,
Copyright © 2010 by Applied Nutriceuticals®
The Effects of a Dietary Supplement, Free Test™, on Serum Free and Total Testosterone Levels in Weight-Trained Male Subjects
Tanis, D., Orrell, D. and Long, Dr. W.
Lab Corp® and Applied Nutriceuticals® Research, Charlotte, North Carolina 28269
Address all correspondence to: Dirk Tanis, Jr., MSci, Department of Research, Applied Nutriceuticals®, 8112 Statesville Road Suite G, Charlotte, NC 28269.
The objective of many weight-trained men is to raise circulating testosterone levels, both through training, and supplementation. In light of these facts, a dietary supplement formulation, Free Test™, was created, combining 3,7-Keto DHEA, Acetyl-L-Carnitine, N-Acetyl-Cysteine, Coleus Forskolii (Forslean®), Resveratrol, Quercetin Dihydrate, B-Vitamins, Magnesium, Zinc, Bioperine®, and Selenium. The product focuses on decreasing the homeostatic mechanisms that lower endogenous testosterone production, while at the same time allowing for sustained, safe increases in free and serum testosterone levels. The purpose of this study was to determine the effects of Free Test™ on serum hormone levels in resistance trained males. Methods: 6 men (mean age, 32.5 yr. ± 10.01, range 18–49 yr) with at least 4 years of weight-training experience were studied. Total (TT) and free (FT) testosterone levels were measured via blood work (LabCorp®) on two separate occasions before (Pre) and after (Post) having taken the dietary supplement, with serum and free testosterone being measured on days (d)= Day 0 and Days 21-61 (mean time period 31.6 d ± 14.87, range 21-61 d). One (1) blood draw per analysis period was taken; Two (2) total draws per subject over two separate days, and during the trial period, subjects were given a 4 capsule daily dosage of the supplement. Results: Each of the 6 weight-trained men (100%) had significant increases in free testosterone levels after 21-61 days of ingesting a 4 capsule daily dosage of Free Test™ when compared to baseline values. Average baseline FT was 9.55 pg/mL ± 1.80 (SD). Average FT following administration of the supplement was 16.98 pg/mL± 5.2, a 78% increase in FT (p<0.10) values for the group.
Numerous studies have documented the anabolic effects of testosterone on skeletal muscle, and this has led to a huge increase in dietary supplement products on the current market that claim to raise testosterone levels and improve body composition. Many testosterone-enhancing supplements have been shown to be either ineffective or dangerous, due to various factors. Some of these products do indeed increase testosterone, but many of the most effective products (Anabolic Steroids and Prohormones) in turn have the most unsafe side effects. Prohormones (PH) and AAS are extremely effective in increasing androgen levels, as they directly bind the androgen receptor (AR), which is the most direct way of increasing testosterone levels. However, as aforementioned, these products can also cause some deleterious side effects, and recently the United States FDA has cracked down on the manufacture of this type of products, and has openly and actively pursued product recalls against companies that distribute prohormones. Similarly, many non-PH and AAS-based testosterone boosters also exist on the current market. Unfortunately, many of these products are completely ineffective, and use a hodgepodge of low extraction-percentage herbals, ineffective or unproven anabolic compounds and/or amino acids. Most of these products use a “kitchen sink” formula, and claim that their combination is “scientifically proven” to work. Some herbal components boost testosterone levels, but most of the time, it is only when taken in completely unattainable dosages that would make a normal person extremely sick or make the product outrageously expensive to produce.
Therefore, an opportunity exists to create a product that can provide a safe way to increase testosterone levels, while being compliant with the Dietary Supplement and Health Act (DSHEA) and other current United States regulatory guidelines. The goal of Free Test™ was to create a formula that focuses on decreasing the homeostatic mechanisms that lower endogenous testosterone production, while at the same time allowing for sustained increases in testosterone, and its positive subsequent effects on lean body mass. The formulation is intended to work not by stimulating the androgen receptor (AR) and having direct androgenic activity, but by manipulating factors that can increase or decrease TT and FT in the body. TT is nearly 100% bound by several different factors- 40% is bound by Steroid Hormone Binding Globulin (SHBG, a β-Globulin that binds hormones such as testosterone and estrogen), 40% is bound by albumin, and 17% is bound by other proteins. The remaining testosterone is considered to be FT, or the unbound, biologically active component of testosterone (TST). Once in the blood, FT can be bound by the AR, and converted to dihydrotestosterone (DHT), or converted to estradiol, via the aromatase enzyme (29).
The product formulation seeks to increase FT via several different indirect means:
• Via Improving the Testosterone:Estrogen (T:E) profile and concurrently increasing testosterone levels through using a naturally-occurring aromatase inhibitor (21-22,27)
• Through Increasing Nitric Oxide (NO) and cyclic guanosine monophsophate ( cGMP) levels, which lead to increases in luteinizing hormone (LH), which is a trigger for greater endogenous testosterone production (1-4,15, 27-28).
• Lowering levels of certain cytokines- many cytokines are inflammatory agents that can inhibit endogenous testosterone production by reducing levels of lutenizing hormone (LH) and decreasing testicular function (25-26,33-34)
• Increasing cyclic adenosine monophosphate ( cAMP) levels, which also increase LH levels (14, 16,17)
The purpose of this pilot work is twofold, in that it allows the researchers to establish that the product does indeed increase TT and FT in a directional manner, and can be used safely for varying periods of time (measured via questionnaire).
Six men between the ages of 18 and 49 yr (mean age, 32.5 ± 10.01) were studied. Eight men were initially chosen; however, one patient had to be excluded because of a pre-existing history of thyroid dysfunction, and another had to be excluded due to the use of an anabolic substance 4 weeks prior to the test. No patients had a history of glucocorticoid or anticonvulsant use, diabetes mellitus, gastrointestinal disease, gastrointestinal surgery, acromegaly, malignancy, or any other known metabolic disease. No patient had a history of alcoholism. All subjects were required to have at least 4 years of weight training experience. There was no history of pubertal or adult testosterone deficiency or growth disturbance, no history of delayed puberty, and no history of pituitary disease or deficiency. As aforementioned, subjects who had used various prohormone derivatives or pharmacologic agents such as anabolic steroids within 6 weeks prior to the study were not allowed to participate. All subjects gave written informed consent, and a questionnaire was given to all subjects regarding how well they tolerated the supplement, protocol for the trial, and also a section where any adverse events or symptoms could be reported (29,31,37).
List of Exclusionary Substances
Refer to WADA 2009 Guidelines for entire list (35)
There were two separate days of testing: Baseline Testing Day (Pre), where each subject had TT and FT measured via blood draw, and Testing Day 2 (Post), where subjects had FT and TT measured via blood draw after having received a 4 capsule daily dosage of the product over a varying time interval (Range 21-61 days). The subject questionnaire was returned after the second blood draw for each subject (29,31,37).
Blood Collection and Assay Methods
Blood samples were taken from an antecubital vein into a 10 ml collection tube, with a 2 mL sample. The samples were handled via Lab Corp® protocol 140103, being allowed to stand at room temperature for 10 min and then centrifuged. The serum was removed and frozen for later analysis. Measured via Lab Corp® protocol 140103 Testosterone,Free (Direct), Serum with Total Testosterone. Routine serum biochemical measurements were made using standard techniques. FT was measured by direct analog/ radioimunnoassay (RIA), and TT was measured by Electrochemiluminescence Immunoassay (ECLIA). FT values were measured in pg/mL; TT values were measured in ng/dL (36).
Subjects were randomly assigned differing lengths of trial supplementation for the product. Two subjects were assigned a 20-22 day trial, three subjects were assigned a 28-30 day trial, and one subject was assigned a 60-62 day trial. The reasoning behind the variable length trials between subject groups was to see if the formulation was sound for elevations in TST over longer, medium, and shorter duration, and to get an idea of whether or not the supplement could be used safely and effectively over varying periods of time. Subjects were instructed to orally ingest 4 capsules upon waking in the AM, preferably with food. During the supplementation period, the physical activity and diet of the subjects was not monitored. Instead, each participant was instructed not to change any of their dietary habits and physical training regime during the trial period (18-19,29,31,37).
Reported Side Effects from Trial
After completing their final blood draw, subjects reported by questionnaire any issues they may have had with the supplementation or the protocol. Subjects were also asked to list any kind of medical side effects that might have occurred during the research.
Due to the nature of pilot research (low number of subjects and only two variables measured)- the design of the study is a quasi-experimental One Group Pretest-Posttest Design. Therefore, a t-test paired for pretest-posttest scores for both TT and FT was utilized. All statistical procedures were performed using GraphPad software and a probability level of p< 0.10 was adopted throughout the study (37).