Fig 1.
A, Fluorescence microscopy images of YFP-tagged AR and mCherry-NLS-mCherry nuclear marker expressed in the reporter cells grown in androgen-depleted cell culture media (upper panels). Incubation with an androgen redistributes the AR to the cell nucleus (lower panels). B, AR BioAssay standard curve: the concentration of T in the media dictates the level of ‘green’ fluorescence of YFP-tagged AR in image regions defined by red fluorescence emitted from mCherry FP-marked cell nuclei change. Maximum AR-YFP nuclear fluorescence is set as 100% and minimum is set as 0%. The nuclear AR level following incubation with a glucuronidase-treated urine sample defines the amounts of androgens present in the well (blue lines), which then is corrected for the urine dilution into the cell culture media to obtain the ‘T-equivalent’ concentration of urinary androgens present.
Fig 2.
Equivalency of androgen (AR BioAssay) and T (MS) measurements in urine of T-treated patients.
AR BioAssay reporter cells were grown in 39.5 μl androgen-depleted culture media and 0.5 μl of glucuronidase-treated urine. The AR-YFP fluorescence level activated by androgens in the urine was compared against the T standard curve to extrapolate androgen concentration in T-equivalent activity levels. That concentration was multiplied by the 80-fold dilution of the urine in the cell culture media. These urine androgen levels (x-axis) then were compared to T measurements conducted by MS on the same samples (y-axis). For T-supplemented individuals, linear regression would be hypothesized to show equivalency (slope = 1.0) of androgen and T levels.
Fig 3.
Rise and fall in urine androgens following injection with T.
Androgen levels measured by AR BioAssay (solid line) and T levels measured by MS in glucuronidase-treated urine from two hypogonadal patients (A and B) pre- and post-T injection (arrows). Samples were collected and stored at home and returned at the next physician visit.
Fig 4.
Efficacy of A, natural steroids and B, AASs on the AR BioAssay. Maximal AR BioAssay activity was determined from dose response curves (Fig 2). The compounds that achieved a maximal level of BioAssay activity at highest concentrations studied (10-6M) are shown in blue; steroids with efficacies comparable to testosterone (100% efficacy) are shown in dark blue. Those with lower efficacies are shown in light blue. Those compounds not achieving a maximum level of BioAssay activity are shown in grey. The mean ± sd for each compound were averaged from the number of independent dose response studies shown (see S1 Fig, which also includes the names and chemical structures of the compounds). In A, compounds are grouped according to global structural similarities to androgens (A), progestagens (P), corticosteroids (C) and estrogens (E).
Fig 5.
Potency (Log EC50) of A, natural steroids and B, AASs in the AR BioAssay. Potency can be established only for compounds that reached maximal activity at the highest concentrations investigated (10-6M). The more negative a Log EC50, the lower the concentration of compound required to reach half-maximal activity (i.e. higher potency). Many of the AAS’s have a higher potency than testosterone. Note that designer AAS’s, such as tetrahydrogestrinone (‘the clear’) are detected in this assay with an efficacy similar to DHT. See Fig 4 for description of symbols and procedures.
Fig 6.
A, Efficacy and B, Potency of AAS metabolites. The metabolites (white bars; e.g. m27) are shown relative to the AASs from which they are metabolized in the body (black bars; e.g. AAS27). See Fig 4 for description of symbols and procedures.
Table 1.
AR BioAssay detection limits for each compound in urine.
Compounds showing any activity are in Bold. Detection limit is set to be AR nuclear fluorescence level of 10% the maximum of the T curve with a dilution of 1 μl of urine into 40 μl of total assay volume. Chemical structures and # of independent studies conducted for each compound are listed in S1 Fig.