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The half maximum inhibitory concentration (IC 50 ) of TEB or TRI to inhibit 5 α -Red1 was determined by adding 1000 nM of testosterone with  mM NADPH and 10 −8 –10 −4  M TEB or TRI in 250  μ l phosphate-buffered saline ( mM) containing 5 α -Red1 protein and incubating each reaction mixture for 60 min. The IC 50 value of TRI to inhibit 3 α -HSD was determined by adding 1000 nM of dihydrotestosterone with  mM NADPH and 10 −8 –10 −4  M TRI in 250  μ l phosphate-buffered saline ( mM) containing 3 α -HSD protein and incubating each reaction mixture for 60 min. For determining the mode of inhibition of 5 α -Red1, 10 −9 –10 −5  M testosterone was added to the reaction mixture in the presence of TEB (10 and 20  µ M) or TRI (20 and 40  µ M) for 5 α -Red1. For determining the mode of inhibition of 3 α -HSD, 10 −9 –10 −5  M dihydrotestosterone was added to the reaction mixture in the presence of TRI (25 and 50  µ M) for 3 α -HSD.

Contraindications
While there has been no definite information published as to who should not take pregnenolone, on theoretical grounds, a few cautions can be suggested. Since pregnenolone (especially at high doses) may (in some people) increase estrogen or testosterone levels, I believe that men with prostate cancer (which may be worsened by testosterone) and women with breast or ovarian cancer (which may be worsened by estrogen) should probably take pregnenolone only with their doctors consent and supervision. Men with high PSA (prostate specific antigen) blood levels (possible indicator for undiagnosed or future prostate cancer) should also proceed with caution with pregnenolone use. Lastly, because of pregnenolones anti-GABA, pro-NMDA action, persons known to suffer from epileptic seizures or who are taking an anti-seizure medication such as Dilantin, Depakote or Tegretol should probably only use pregnenolone with their doctors supervision. Finally, as we age, the body produces ever-less of the enzyme which converts pregnenolone to DHEA. Thus, while supplementary pregnenolone taken during middle age and beyond will produce at least some normalization back toward more youthful (and healthful) levels of other steroid hormones, pregnenolone will not completely substitute for other steroid hormone supplements in those with medically demonstrated needs for various specific steroids ., DHEA, cortisol, estrogen, etc.
 

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