Unauthorized ingredients in "nootropic" dietary supplements: A review of the history, pharmacology, prevalence, international regulations, and potential as doping agents.

The first nootropic prohibited in sport was fonturacetam (4-phenylpiracetam, carphedon) in 1998. Presented here 25 years later is a broad-scale consideration of the history, pharmacology, prevalence, regulations, and doping potential of nootropics viewed through a lens of 50 selected dietary supplements (DS) marketed as "cognitive enhancement," "brain health," "brain boosters," or "nootropics," with a focus on unauthorized ingredients. Nootropic DS have risen to prominence over the last decade often as multicomponent formulations of bioactive ingredients presenting compelling pharmacological questions and potential public health concerns. Many popular nootropics are unauthorized food or DS ingredients according to the European Commission including huperzine A, yohimbine, and dimethylaminoethanol; unapproved pharmaceuticals like phenibut or emoxypine (mexidol); previously registered drugs like meclofenoxate or reserpine; EU authorized pharmaceuticals like piracetam or vinpocetine; infamous doping agents like methylhexaneamine or dimethylbutylamine; and other investigational substances and peptides. Several are authorized DS ingredients in the United States resulting in significant global variability as to what qualifies as a legal nootropic. Prohibited stimulants or ß2-agonists commonly used in "pre-workout," "weight loss," or "thermogenic" DS such as octodrine, hordenine, or higenamine are often stacked with nootropic substances. While stimulants and ß2-agonists are defined as doping agents by the World Anti-Doping Agency (WADA), many nootropics are not, although some may qualify as non-approved substances or related substances under catch-all language in the WADA Prohibited List. Synergistic combinations, excessive dosing, or recently researched pharmacology may justify listing certain nootropics as doping agents or warrant additional attention in future regulations.

The influence of diet on isotope ratio mass spectrometry values.

OBJECTIVES: Athletes have increasingly used testosterone (T) and other endogenous anabolic steroids that cannot be detected by conventional gas chromatography-mass spectrometry. This led to gas chromatography-combustion-isotope ratio mass spectrometry(GC/C/IRMS), which measures the relative amount of 13C in urinary steroids. Because exogenous testosterone is relatively low in 13C content, this study will determine if consuming a diet low in 13C plants, such as soy, can be confused with a GC/C/IRMS-positive test for exogenous testosterone. DESIGN: Cross-sectional study in which 22 vegetarians known to consume a diet depleted of 13C isotope were compared with a geographic control group of 14 subjects consuming a normal diet. SETTING: Two distinct subject populations with respect to diet. SUBJECTS: Subjects were recruited from a soy-based cooperative and control volunteers. Twenty-two of 24 research subjects completed the protocol compared with 14 of 22 control subjects. INTERVENTIONS: Independent variables were delta13C IRMS values,urinary steroid profile, and isoflavone analysis. MAIN OUTCOME MEASURES: Comparisons were made with respect to dietary analysis, isoflavones, and urinary steroid measurements using GC-C-IRMS. RESULTS: The delta13C values for 2 major metabolites of T (androsterone and etiocholanolone) were lower for the vegetarians than the controls (P = 0.005). The vegetarians excreted a median of 23 micromol/d of total isoflavones compared with 2.7 micromol/d for the control group (P =0.0002). CONCLUSIONS: The carbon isotope ratios of urinary testosterone metabolites of vegetarians consuming a diet that is markedly depleted of 13C content were lower than that of control subjects, but not low enough to result in World Anti-Doping Agency criteria for a positive IRMS analysis.

Low-fat high-fiber diet decreased serum and urine androgens in men.

To validate our hypothesis that reduction in dietary fat may result in changes in androgen metabolism, 39 middle-aged, white, healthy men (50-60 yr of age) were studied while they were consuming their usual high-fat, low-fiber diet and after 8 wk modulation to an isocaloric low-fat, high-fiber diet. Mean body weight decreased by 1 kg, whereas total caloric intake, energy expenditure, and activity index were not changed. After diet modulation, mean serum testosterone (T) concentration fell (P < 0.0001), accompanied by small but significant decreases in serum free T (P = 0.0045), 5 alpha-dihydrotestosterone (P = 0.0053), and adrenal androgens (androstendione, P = 0.0135; dehydroepiandrosterone sulfate, P = 0.0011). Serum estradiol and SHBG showed smaller decreases. Parallel decreases in urinary excretion of some testicular and adrenal androgens were demonstrated. Metabolic clearance rates of T were not changed, and production rates for T showed a downward trend while on low-fat diet modulation. We conclude that reduction in dietary fat intake (and increase in fiber) results in 12% consistent lowering of circulating androgen levels without changing the clearance.

Another designer steroid: discovery, synthesis, and detection of 'madol' in urine.

Madol (17alpha-methyl-5alpha-androst-2-en-17beta-ol) was identified in an oily product received by our laboratory in the context of our investigations of designer steroids. The product allegedly contained an anabolic steroid not screened for in routine sport doping control urine tests. Madol was synthesized by Grignard methylation of 5alpha-androst-2-en-17-one and characterized by mass spectrometry and NMR spectroscopy. We developed a method for rapid screening of urine samples by gas chromatography/mass spectrometry (GC/MS) of trimethylsilylated madol (monitoring m/z 143, 270, and 345). A baboon administration study showed that madol and a metabolite are excreted in urine. In vitro incubation with human liver microsomes yielded the same metabolite. Madol is only the third steroid never commercially marketed to be found in the context of performance-enhancing drugs in sports.

Tetrahydrogestrinone: discovery, synthesis, and detection in urine.

Tetrahydrogestrinone (18a-homo-pregna-4,9,11-trien-17beta-ol-3-one or THG) was identified in the residue of a spent syringe that had allegedly contained an anabolic steroid undetectable by sport doping control urine tests. THG was synthesized by hydrogenation of gestrinone and characterized by mass spectrometry and NMR spectroscopy. We developed and evaluated sensitive and specific methods for rapid screening of urine samples by liquid chromatography/tandem mass spectrometry (LC/MS/MS) of underivatized THG (using transitions m/z 313 to 241 and 313 to 159) and gas chromatography/high-resolution mass spectrometry (GC/HRMS) analysis of the combination trimethylsilyl ether-oxime derivative of THG (using fragments m/z 240.14, 254.15, 267.16, and 294.19). A baboon administration study showed that THG is excreted in urine.

Testosterone metabolic clearance and production rates determined by stable isotope dilution/tandem mass spectrometry in normal men: influence of ethnicity and age.

The metabolic clearance rate (MCRT) and production rate (PRT) of testosterone (T) were measured using constant infusion of trideuterated (d3) T and quantitating serum d3T by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Serum unlabeled T (d0T) was measured by LC-MS-MS, and serum total T (d3T + d0T) was measured by RIA. Mean MCRR (measured by LC-MS-MS) in young white men (1272 +/- 168 liters/d) was not significantly different from young Asian men (1070 +/- 166 liters/d). Mean PRT was also not significantly different between the two ethnic groups (whites, 9.11 +/- 1.11 mg/d; Asians, 7.22 +/- 1.15 mg/d; P = 0.19 using d0T data). Both the mean MCRR (812 +/- 64 liters/d; P < 0.01) and the PRT (3.88 +/- 0.27 mg/d; P < 0.001) were significantly lower in middle-aged white men when compared with their younger counterparts. The mean MCRR and PRR calculated using serum total T or d0T data showed a diurnal variation, with levels at midday significantly higher than those measured in the evening in the young (MCRT, P < 0.01; PRT, P < 0.001) and to a lesser extent in the older men (MCRT, P < 0.05; PRT, P < 0.05 using total T and P < 0.001 using d0T data). We conclude that using LC-MS-MS to detect d3T in serum after constant infusion of stable isotope-labeled T allows the measurements of MCRT and PRT, which can be used to study androgen metabolism repeatedly after physiological or pharmacological interventions.

Measurement of total serum testosterone in adult men: comparison of current laboratory methods versus liquid chromatography-tandem mass spectrometry.

The diagnosis of male hypogonadism requires the demonstration of a low serum testosterone (T) level. We examined serum T levels in pedigreed samples taken from 62 eugonadal and 60 hypogonadal males by four commonly used automated immunoassay instruments (Roche Elecsys, Bayer Centaur, Ortho Vitros ECi and DPC Immulite 2000) and two manual immunoassay methods (DPC-RIA, a coated tube commercial kit, and HUMC-RIA, a research laboratory assay) and compared results with measurements performed by liquid chromatography-tandem mass spectrometry (LC-MSMS). Deming's regression analyses comparing each of the test results with LC-MSMS showed slopes that were between 0.881 and 1.217. The interclass correlation coefficients were between 0.92 and 0.97 for all methods. Compared with the serum T concentrations measured by LC-MSMS, the DPC Immulite results were biased toward lower values (mean difference, -90 +/- 9 ng/dl) whereas the Bayer Centaur data were biased toward higher values (mean difference, +99 +/- 11 ng/dl) over a wide range of serum T levels. At low serum T concentrations (<100 ng/dl or 3.47 nmol/liter), HUMC-RIA overestimated serum T, Ortho Vitros ECi underestimated the serum T concentration, whereas the other two methods (DPC-RIA and Roche Elecsys) showed differences in both directions compared with LC-MSMS. Over 60% of the samples (with T levels within the adult male range) measured by most automated and manual methods were within +/- 20% of those reported by LC-MSMS. These immunoassays are capable of distinguishing eugonadal from hypogonadal males if adult male reference ranges have been established in each individual laboratory. The lack of precision and accuracy, together with bias of the immunoassay methods at low serum T concentrations, suggests that the current methods cannot be used to accurately measure T in females or serum from prepubertal subjects.

Liquid chromatography-tandem mass spectrometry assay for human serum testosterone and trideuterated testosterone.

A liquid chromatography tandem mass spectrometry assay for serum testosterone (T) and trideuterated testosterone (d(3)T) was developed in order to support clinical research studies that determine the pharmacokinetics, production rate, and clearance of testosterone by administration of trideuterated testosterone. After adding 19-nortestosterone as the internal standard (I.S.), sodium acetate buffer, and ether, to a serum aliquot, the mixture was shaken and centrifuged, and the ether was dried. The extract was reconstituted in methanol and 15 microl was injected into a liquid chromatograph equipped with an autosampler and Applied Biosystems-Sciex API 300 triple quadrupole mass spectrometer operated in the positive ion mode. T, d(3)T, and I.S. were monitored with transitions m/z 289 to m/z 97, m/z 292 to m/z 97, and m/z 275 to m/z 109, respectively. The two calibration curves were linear over the entire measurement range of 0-20 ng/ml for T and 0-2.0 ng/ml for d(3)T. The LOQs for T and d(3)T were 0.5 ng/ml and 0.05 ng/ml. The recoveries for T and d(3)T were 91.5 and 96.4%. For T at 1.25 ng/ml and 4.0 ng/ml, the intra-day precision (RSD, %) was 3.9 and 4.3% and intra-day accuracy 0.01 and 4.5%, respectively. The inter-day precision at these levels was 5.3 and 5.4% and inter-day accuracy was 1.9 and 0.3%. For d(3)T at 0.125 ng/ml and 0.4 ng/ml, the intra-day precision (RSD, %) was 2.8 and 8.3% and intra-day accuracy was 1.8 and 5.6%. The inter-day precision at these levels was 10.0 and 7.6% and inter-day accuracy was 5.7 and 3.4%. The concentrations of T in the 38 healthy subjects ranged from 2.5 to 14.0 ng/ml (mean 6.2 ng/ml).

Detection of recombinant human erythropoietin in urine by isoelectric focusing.

BACKGROUND: Doping with erythropoietic proteins such as recombinant human erythropoietin (rHuEPO) and darbepoetin alfa is a serious issue in sport. There is little information on the time course of detection of rHuEPO in urine and on methods to evaluate electrophoresis-based data. METHODS: We used a recently described isoelectric focusing method for detecting rHuEPO and endogenous EPO in urine obtained from individuals treated with placebo or epoetin alfa. The latter was administered subcutaneously at 50 IU/kg on days 0, 2, 4, 7, 9, 11, 14, 16, and 18. Blood and urine samples were collected during the morning of study days -3, 0, 2, 4, 7, 9, 11, 14, 16, and 18 and on days 2, 3, 4, and 7 postadministration. We developed visual and numerical (two-band ratio) techniques to evaluate the electropherograms for the presence of rHuEPO. RESULTS: Compared with the placebo group, the epoetin alfa-treated group responded with increases in hematocrit, reticulocytes, macrocytes, serum EPO, and serum soluble transferrin receptor. The electropherograms showed that the pattern of bands arising from urinary rHuEPO is different from that of endogenous urinary EPO. Both the two-band ratio and the visual technique detected rHuEPO in all 14 epoetin alfa-treated individuals 3 days after the last dose. On the 7th day after the last dose, both techniques detected rHuEPO in approximately one-half of the participants. rHuEPO was not detected in the placebo-treated individuals. CONCLUSIONS: The isoelectric focusing method detects rHuEPO in most urine samples collected 3 days after nine doses of epoetin alfa. The numerical two-band ratio was equivalent to a visual method for detecting rHuEPO in urine.

Effects of oral androstenedione administration on serum testosterone and estradiol levels in postmenopausal women.

Androstenedione is a steroid hormone and an intermediate in the synthetic pathway of both testosterone and estradiol in men and women. It is available without prescription and taken with the expectation that it may have beneficial effects on strength, general well-being, libido, and quality of life. Although studies have shown that oral androstenedione increases serum testosterone and estradiol levels in men, the hormonal effects of androstenedione in postmenopausal women are unknown. We randomly assigned 30 healthy postmenopausal women to receive 0, 50, or 100 mg androstenedione as a single oral dose. After androstenedione administration, we made hourly measurements of serum androstenedione, estrone, estradiol, and testosterone concentrations during 12 h of frequent blood sampling. The mean change (+/-SD) in serum androstenedione area under the curve (AUC) was greater in both the 50-mg (79 +/- 39%) and 100-mg dose groups (242 +/- 184%) than in the control group (-29 +/- 28%) (P < 0.0001 for controls vs. 50-mg group and controls vs. 100-mg group). The mean change in serum androstenedione AUC was also greater in the 100-mg than 50-mg dose group (P = 0.0026). The mean change in serum estrone AUC was greater in both the 50-mg (108 +/- 72%) and 100-mg dose groups (116 +/- 119%) than in the control group (-5 +/- 19%), although the control vs. 100-mg group comparison did not quite meet statistical significance (P < 0.0001 for controls vs. 50-mg group, P = 0.0631 controls vs. 100-mg group). The mean change in serum estradiol AUC remained stable after supplementation in all groups without any between-group differences observed (-11 +/- 17%, 2.8 +/- 34%, -11 +/- 27%, for the control, 50-mg, and 100-mg groups, respectively). The mean change in serum testosterone AUC was greater in both the 50-mg (185 +/- 146%) and 100-mg dose groups (457 +/- 601%) than in the control group (-27 +/- 13%) (P < 0.0001 for controls vs. 50-mg group and for controls vs. 100-mg group). The mean change in testosterone AUC was also greater in the 100-mg dose group than 50-mg dose group (P = 0.0257). There was considerable individual variability in the changes of serum androstenedione, estrone, and testosterone levels in the treated groups with peak serum testosterone levels exceeding the upper limit of normal in 4 of 10 women in the 50-mg dose group and 6 of 10 in the 100-mg dose group. We concluded that the acute administration of both 50-mg and 100-mg of androstenedione increases serum testosterone and estrone levels, but not estradiol levels, in postmenopausal women. If these hormonal effects are sustained during long-term administration, regular use of this supplement by postmenopausal women could thus cause both beneficial and adverse effects.

Detection of norbolethone, an anabolic steroid never marketed, in athletes' urine.

Norbolethone (13-ethyl-17-hydroxy-18,19-dinor-17alpha-pregn-4-en-3-one) is a 19-nor anabolic steroid first synthesized in 1966. During the 1960s it was administered to humans in efficacy studies concerned with short stature and underweight conditions. It has never been reported by doping control laboratories. Norbolethone was identified in two urine samples from one athlete by matching the mass spectra and chromatographic retention times with those of a reference standard. The samples also contained at least one likely metabolite. The samples were also unusual because the concentrations of endogenous steroids were exceptionally low. Since norbolethone is not known to be marketed by any pharmaceutical company, a clandestine source of norbolethone may exist.

Effects of androstenedione administration on epitestosterone metabolism in men.

Androstenedione is a steroid hormone sold over-the-counter to individuals who expect that it will enhance strength and athletic performance. Endogenous androstenedione is the immediate precursor of testosterone. To evaluate the metabolism of oral androstenedione, we randomly assigned 37 healthy men to receive 0 (group 1), 100 mg (group 2), or 300 mg (group 3) of androstenedione in a single daily dose for 7 days. Eight-hour urines were collected 1 day before the start of androstenedione, and on days 1 and 7. Using gas chromatography-mass spectrometry, we measured excretion rates of glucuronide-conjugated epitestosterone, its putative precursor (E-precursor), and metabolites (EM-1 and EM-2), and we evaluated possible markers of androstenedione administration. Day 1 and 7 rates were not different: the means were averaged. The means (microg/h) for groups 1, 2, and 3, respectively were, for epitestosterone 2.27, 7.74, and 18.0; for E-precursor, 2.9, 2.0, and 1.5; for EM-1/E-precursor 0.31, 1.25, and 2.88; for EM-2/E-precursor 0.14, 0.15, and 1.15; for testosterone/epitestosterone (T/E) 1.1, 3.5, and 3.2. Epitestosterone, EM-1, and EM-2 excretion was greater in groups 2 and 3 versus group 1 (0.0001 < P < 0.03), as were EM-1/E-precursor, EM-2/E-precursor, and T/E. E-precursor excretion was lower in groups 2 (P = 0.08) and 3 (P = 0.047) versus group 1. Androstenedione increases excretion of epitestosterone and its two metabolites, while decreasing that of its precursor. Elevated ratios of EM-1- and EM-2/E-precursor, and the presence of 6alpha-hydroxyandrostenedione are androstenedione administration markers.

Detection of epitestosterone doping by isotope ratio mass spectrometry.

BACKGROUND: Epitestosterone is prohibited by sport authorities because its administration will lower the urinary testosterone/epitestosterone ratio, a marker of testosterone administration. A definitive method for detecting epitestosterone administration is needed. METHODS: We developed a gas chromatography-combustion-isotope ratio mass spectrometry method for measuring the delta(13)C values for urinary epitestosterone. Sample preparation included deconjugation with beta-glucuronidase, solid-phase extraction, and semipreparative HPLC. Epitestosterone concentrations were determined by gas chromatography-mass spectrometry for urines obtained from a control group of 456 healthy males. Epitestosterone delta(13)C values were determined for 43 control urines with epitestosterone concentrations > or =40 microg/L (139 nmol/L) and 10 athletes' urines with epitestosterone concentrations > or =180 microg/L (624 nmol/L), respectively. RESULTS: The log epitestosterone concentration distribution was gaussian [mean, 3.30; SD, 0.706; geometric mean, 27.0 microg/L (93.6 nmol/L)]. The delta(13)C values for four synthetic epitestosterones were low (less than or equal to -30.3 per thousand) and differed significantly (P <0.0001). The SDs of between-assay precision studies were low (< or =0.73 per thousand). The mean delta(13)C values for urine samples obtained from 43 healthy males was -23.8 per thousand (SD, 0.93 per thousand). Nine of 10 athletes' urine samples with epitestosterone concentrations >180 microg/L (624 nmol/L) had delta(13)C values within +/- 3 SD of the control group. The delta(13)C value of epitestosterone in one sample was -32.6 per thousand (z-score, 9.4), suggesting that epitestosterone was administered. In addition, the likelihood of simultaneous testosterone administration was supported by low delta(13)C values for androsterone and etiocholanolone. CONCLUSIONS: Determining delta(13)C values for urinary epitestosterone is useful for detecting cases of epitestosterone administration because the mean delta(13)C values for a control group is high (-23.8 per thousand) compared with the delta(13)C values for synthetic epitestosterones.

Assessing the Threat of Anabolic Steroids.

Sprinter Ben Johnson's lifelong expulsion from international competition forces the sports medicine community to acknowledge, once again, the temptation performance-enhancing drugs pose for athletes. The drive to jump a little higher, run a little faster, or be a little stronger continues to compel some athletes to seek an edge through drugs. To better prepare physicians who must guide patients faced with these temptations, we gathered four medical experts to discuss current information about anabolic-androgenic steroids.