Confusing Study Results: Saliva Testing "Adequate" For Free Testosterone, Yet Way Off For Cortisol!?

Many of us spend a fortune on "hormone optimization", but when it comes to actually testing our levels, most of us are pretty cheap: "200$ for labs? No way I rather buy another test booster for that money - won't hurt, I guess!" And even, if we finally decide to test our testosterone levels, we will usually choose the cheapest and most convenient testing method: saliva testing. But is this an adequate way of assessing one's hormonal status? Although these tests have been around forever, nobody appears to be sure how reliable they actually are. A recent study (Caenegem. 2011) from the University Hospital of Ghent, Belgium, attempts to provide answers.

The researchers tested two saliva collection methods, chewing on a cotton swab (Salivette, Sarstedt) and passive drooling on 30 healthy males with a median age of 27 years (range 19–65 years) and compared their findings against serum levels, which were measured on the same occasion. Here are their results:

For cortisol, we observed a mean positive bias of 65% for passive drooling versus Salivette. In addition, cortisol determined on passive drooling samples correlated much worse with calculated free cortisol than cortisol analyzed on the cotton samples (respectively r=0.34 versus r=0.70). However, for testosterone passive drooling correlated well with the calculated free fraction (r=0.66). A positive bias of 21% for testosterone collected by passive drooling versus Salivette was observed. In contrast to earlier reports, we found no artefactually high testosterone results with the cotton-based collection method.

Obviously, these results seem to indicate that saliva testing for testosterone is a good, while saliva testing for cortisol is a bad idea, BUT these results are in fact intrinsically flawed. By just calculating free serum values instead of using equilibrium dialysis to actually measure them, the scientists' control values and with them their assessment of the "appropriateness" and "inappropriateness" of salivary testosterone, respectively cortisol testing are questionable. 

From what I hear from practitioners like Dr Crisler, it is highly advisable to spend the extra cash to have measured (not calculated) serum testosterone levels as reliable baseline and 4x saliva cortisol profiles to see where your levels are at throughout a 24h period. Arguably, this is yet another case in which a questionable study design leads to results which refute common practice and aggravate a confusion around hormonal testing procedures which affects practitioners and patients, alike.

5α-androstane-3,6,17-trione - Kneller's Trione: Newly Patented Aromatase Inhibitor that Raises Testosterone Without Lowering Estrogen Levels!?

I happened onto an interesting patent (Kneller. 2011), published on 02/10/2011 the author of which, Bruce Kneller, who was arrested in the course of a steroid bust back in 2006, claims to have invented an aromatase inhibitor, 5α-androstane-3,6,17-trione (Kneller's Trione) which produces "substantial and significant increases in plasma testosterone levels in men while inducing no changes outside the accepted normal limits for plasma estradiol levels". This is very different from your usual AI (ATD, Arimidex, Letrozole, etc.) which tend to eradicate estrogen and thus produce nasty side effects. As far as its concrete applications are concerned the author writes:

The oral, daily dose of Kneller's Trione can be from about 25 mg to about 750 mg per day, such as, for example, from about 25 mg to about 500 mg per day, from about 25 mg to about 250 mg per day, or from about 25 to about 100 mg per day. [...] In some embodiments of the invention the composition comprising Kneller's Trione is formulated as a tablet, capsule, caplet, powder, suspension, gel preparation, aqueous solution, solid food form (e.g., chewable bar or wafer), or liquid dosage form such as elixirs, syrups, dispersed powders, granules or emulsions.

Bruce W. Kneller then cites a pilot study in which Kneller's Trione was administered orally to three individuals at dosages of 25mg, 50mg and 100mg.

Figure 1: Tables from patent of Kneller's Trione

As can be seen from the tables in figure 1 the effect of 5α-androstane-3,6,17-trione depends on a) previous hormone levels and b) dosage. Most interestingly administration of 50mg Kneller's Trione to subject 2 who had low estradiol levels to begin with did not reduce these levels any further. With reference to the optimal dosing scheme and potential side effects Kneller reports:

Although dosing of Kneller's Trione went as high as 750 mg per day in this pilot study, no added benefits were seen with dosages this high. Dosages of 250 mg and 500 mg per day also raised total testosterone and bioavailable testosterone levels substantially and safely but did not seem to offer any added benefit over a dose of 100 mg per day. At the 750 mg per day oral dosing level, occurrence of a priaprism caused the subject to withdraw from participation in this pilot study. No other adverse events were noted by any study subject during this pilot study. Observations of every subject's liver function, kidney function, blood lipid levels, blood pressure, heart rate, respirations, and body temperature were made at frequent intervals during this pilot study and were found to be within medically established normal ranges and values.

While my cursory web-search for available OTC supplements with this product did not produce any results, I assume it won't take long until we see a range of these products on the market - even in view of the fact that a constant boner (priapism) may not be the worst, but certainly an annoying and in the long run painful side effect ;-)

As always, I will inform you as soon as there are more recent study results and or product releases. In view of the fact, that Kneller is also the patent-holder of the "OUTLAST" formula in Gaspari Nutrition's cell-volumizer SizeON it's most likely that we will see a "new Novedex XT" hit the market soon.

Edit: Kevin just posted a comment mentioning, he believes that it is already in "some Gaspari products" and in fact. He is right! Its part of Gaspari's Halodrol MT, which I believe is not produced anymore, though. Well, I assume that's what happens to things which work, these days...

Even Low "Normal" Plasma Testosterone Levels Associated with 2.5x Higher Risk of Insulin Resistance

"Your levels are in range!" Did you ever have your doctor say that to you? It appears as if next time you hear that sentence, you better ask him "Where 'in range' are my levels, exactly?" At least, the results of a recently published study by Menendez et al. (Menendez. 2011) suggests that it might be worth digging somewhat deeper into this issue. Studying a cohort of 282 men (aged 36 to 85 years) who had "normal concentrations of total testosterone", the scientists found:

Serum concentrations of testosterone and bioavailable testosterone were negatively correlated with age, body mass index, waist circumference, blood glucose, glycated hemoglobin levels and insulin. Serum concentrations of total testosterone, bioavailable testosterone and SHBG were lower in men with glucose intolerance or diabetes than in those with normal glucose tolerance. After multivariate analysis, age and total testosterone levels were independent predictors of the presence of diabetes or glucose intolerance. The risk of glucose intolerance or diabetes mellitus was over 2.5 times higher in men with total testosterone levels in the lowest quartile than in those with total testosterone in the top quartile.

While the results of this study are certainly interesting and significant, it is unfortunately impossible to tell, whether glucose intolerance and diabetes reduce the natural production of testosterone, OR low testosterone levels reduce glucose intolerance and lead to diabetes. In other words: What comes first, low testosterone or glucose intolerance? Further research will be needed to answer this question, but if you want my opinion, I suspect that they just go hand in hand, i.e. a bad diet lowers glucose tolerance, high sugar reduces testosterone production, low androgen levels impair glucose tolerance etc. - a viscous circle.

News on USP Labs' "Pink Magic": High Dose Massularia Acuminata Increases Testosterone and Makes Bucks Horny as Hell

Faithful readers of the SuppVersity will probably remember my short write-up on USP Labs Test Booster "Pink Magic". Now that the initial fuss about how "brilliant" and "unique" the product is, is over there is finally another study on the efficiency of its main ingredient, a shrub called massularia acuminata.

Two scientists from Nigeria (Yakubo. 2011) published an article on the "Effect of Aqueous Extract of Massularia acuminata Stem on Sexual Behaviour of Male Wistar Rats" in the January issue of Evidence Based Complementary Alternative Medicine. The scientists found that supplementation with 500 and 1000mg/kg body weight (equivalent to 6.8g and 13.6g) of an aqueous extract of the stem of the botanical...

... increased the frequencies of mount and intromission. In addition, the ejaculation latency was significantly prolonged (P < .05). The latencies of mount and intromission were reduced significantly whereas ejaculation frequency increased. The extract also reduced the postejaculatory interval of the animals. Computed percentages of index of libido, mounted, intromitted, ejaculated and copulatory efficiency were higher in the extract treated animals compared to the distilled water-administered control whereas the intercopulatory interval decreased significantly.

In short: the rats were horny as hell ;-) But there was more to it:

The extract also significantly (P < .05) increased the serum testosterone content of the animals except in those administered with 250mg/kg body weight on days 1 and 3.

In view of these results it is interesting to have a look at the amount of massularia in Pink Magic. Unfortunately, USP provides no data on the amount of the individual ingredients - so, we only know that there is 1.6g of a mixture of massularia and two other botanicals in one serving. Massularia is mentioned first which tells us that it should be the main ingredient. So, let's be generous and assume that there is 1.0g of massularia per serving. USP recommends 3 servings per day. This would amount to 3.0g of massularia, which has now been clinically proven to be insufficient; and what's more even with 6.8g/day (equivalent to 500mg/kg in rats) the increase in testosterone (cf. Table 1) is still almost negligible.

Table 1: Effect of aqueous extract of Massularia acuminata stem on serum testosterone concentrations of male rats. (Yakubo. 2011)

What about all the positive feedback on various message boards, then? Well, we do not know how the dosing protocol used in rats eventually translates to human beings. The above calculation is based on a standard human equivalence dose formula, the accuracy of which is certainly questionable. Thus, the results of the study are actually good news for USP Labs, as they prove that massularia which, by the way is not a USP Labs exclusive anymore, actually works.

Vitamin D Supplementation(!) Increases Testosterone in Men

Thanks to Dashforce from the MindAndMuscle-Forum we finally have evidence for the direct effect of vitamin D3 supplementation on testosterone in men. Dashforce reports on a study by scientists from Austria and Germany (Pilz. 2010) who provided 54 men, whose serum levels were previously at the lower range ~30ng/ml (the referential  minimum varies depending on which source you cite from 30-50ng/ml),with either 83 μg (3 332 IU) vitamin D daily or placebo for 1 year.

Table 1: Characteristics of the study groups at baseline and at the end of the study

As you can see in table 1,vitamin D supplementation lead to a significant increase in total testosterone from 10.7 nmol/l to 13.4 nmol/l. What's even more important, this increase (+25%) was not compensated by and an increase of SHGB, so that the amount of free testosterone rose from 0.222 n/mol to 0.267 nmol/l, i.e. by +20% (vs. +5% in the placebo group).

What is particularly interesting about this study is the fact that supplementation actually raised the level of testosterone. This is different from most previous studies, which observed a correlation between higher vitamin D levels and higher testosterone levels. If for example some probands would simply have been more healthy and thus exhibit higher levels of both, we would have had the same correlation. In the case of the Pilz study there is a clear causative effect between restauration of adequate vitamin D levels and increases in total and free testosterone.

I hope we see more trials with a similar design - also to investigate if there is a dose response relationship and whether even higher vitamin D levels would further increase testosterone. Until then, keep an eye on your vitamin D level and take a supplement to get to the upper ranges of the "normal" range ~80ng/ml.