Impact of Biotin Supplementation on Human Chorionic Gonadotropin Immunoassays Utilizing Biotin-Streptavidin Binding Methods in Urine.

BACKGROUND: Human chorionic gonadotropin (hCG) detection is indicative of pregnancy and can be indicative of some forms of cancerous tumors. The hCG drug itself, however, is a performance enhancing substance used by male athletes to increase testosterone production. Antidoping testing for hCG is conducted in urine, often on immunoanalyzer platforms, many of which utilize biotin-streptavidin dependent immunoassays in which the presence of biotin in samples is a known confounding factor. While biotin interference in serum has been well-studied, the extent of biotin interference in urine has not. METHODS: Ten active male individuals underwent a 2-week hCG administration protocol concurrent with supplementation with biotin (20 mg/day) or placebo. Urine and serum samples were collected throughout the study and analyzed for hCG and biotin concentrations. RESULTS: Urinary biotin levels in the hCG + biotin group increased 500-fold over baseline and 29-fold over corresponding serum biotin levels after biotin supplementation. When using a biotin-dependent immunoassay, the hCG + placebo group produced hCG-positive results (hCG ≥ 5 mIU/mL) in 71% of urine samples, while the hCG + biotin group produced positive results in only 19% of samples. Both groups had elevated hCG values in serum measurements by a biotin-dependent immunoassay and in urine when using a biotin-independent immunoassay. Urinary hCG measurements and biotin levels from the hCG + biotin group showed a negative correlation (Spearman r = -0.46, P < 0.0001) when measured using a biotin-dependent immunoassay. CONCLUSIONS: Biotin supplementation can severely suppress urinary hCG values in assays utilizing biotin-streptavidin binding methods and therefore these types of assays are not recommended for use in urine samples containing high levels of biotin. Clinicaltrials.gov Registration Number: NCT05450900.

Is blood blood? Comparing quantitation of endogenous steroids and luteinizing hormone in concurrently collected venous serum and Tasso+ SST capillary serum samples.

The monitoring of endogenous steroids in urine has been an important component of the Athlete Biological Passport (ABP) for the last decade. Recently, the quantitation of endogenous steroids in blood has been incorporated into the ABP to increase sensitivity in circumstances where the excretion of urinary ABP biomarkers is low. Current ABP guidelines mandate the use of venous blood draws for blood steroid sample collections, however, recent efforts have focused on investigating the use of less invasive sample collection methods, such as capillary blood collected from the upper arm. The focus of this study was to compare the analytical results of venous and capillary blood collected weekly from 20 individuals, 10 males and 10 females, over six weeks. The two primary biomarkers of the blood steroid ABP module, testosterone (T) and the testosterone/androstenedione (T/A4) ratio, were compared, as well as luteinizing hormone (LH) and the T/LH ratio in male participants, two biomarkers known to be responsive to T use. All biomarkers showed excellent agreement between venous and capillary blood. Longitudinal stability between sample types within individuals was also comparable for all biomarkers. Finally, storage of simultaneously collected capillary samples at room temperature and frozen conditions was compared with evaluate the potential impact of non-cold chain shipping conditions. Most biomarkers showed excellent agreement between frozen and room temperature storage conditions. These results indicate capillary blood collections represent a promising alternative to venous blood collections for the blood steroid module of the ABP.

Assessing EPO stability in urine and comparing recombinant EPO detectability in matched urine, venous serum, and capillary serum following a controlled epoetin alfa administration.

The instability of erythropoietin receptor agonists (ERAs, i.e., EPO) in urine presents a challenge to their detectability in doping control samples; however, this issue is not often seen in blood (serum) samples. With the anti-doping field beginning to transition into alternative blood collection technologies, it is important to understand recombinant EPO (rEPO) detectability in serum samples collected from one such capillary collection device, the Tasso+ SST. Twelve individuals were administered a single, 40 IU/kg dose of rEPO (epoetin alfa, EPOGEN®). Following administration, matched urine, venous serum, and capillary serum samples were concurrently collected. Urine aliquots were subject to various storage times and temperatures mimicking shipping conditions of doping control urine samples to assess EPO stability, while other urine aliquots, venous serum, and capillary serum aliquots were frozen until analysis to understand rEPO detectability across all three matrices. EPO and rEPO instability was identified in urine collected from 8 of 12 participants, especially in aliquots stored at room temperature and 37°C. In some of these unstable samples, rEPO was still detectable, while in others, no recombinant nor endogenous EPO was detectable and would have resulted in negative sample reports. Analyzing the concurrently collected urine, venous, and capillary serum samples, rEPO detectability was identical across the three matrices. In most cases, rEPO was detectable for at least 168 h post-administration. Noting greater stability in blood compared with urine, it is recommended that anti-doping authorities utilize this novel capillary serum collection technology to improve overall ERA detectability in doping control samples.

Evaluation of Minimum Reporting Limits to Determine In-Competition Use of Stimulants.

The applicability of urinary minimum reporting limits (MRLs) to determine in-competition use of prohibited substances is an evolving topic. Most stimulants are subject to a universal MRL, despite the wide range of commercially available dosages for commonly used stimulants. Further, it is unknown whether the urinary MRL is reflective of a pharmacological dose ingested after the start of the in-competition period. To evaluate whether urinary MRLs can distinguish between in-competition and out-of-competition use, a controlled administration study was performed with three commonly used stimulants-amphetamine, methylphenidate, and modafinil at relatively low but therapeutically relevant dosages. Four to six volunteers were administered a particular drug once per day for five consecutive days. Urine, serum, dried blood spots (DBS), and oral fluid (OF) were collected during the active administration period and for 48 h after cessation of use. For all participants, urinary concentrations for all target analytes exceeded the MRL even 48 h after cessation of use. In serum and DBS, most volunteers showed detectable amounts at 48 h post use. Peak concentrations were variable between target compounds even with similar administered dosages. Further, there was a reproducible difference between serum and DBS concentrations. Interpretation of results from OF measurements was challenging due to the inability to normalize for hydration status and OF viscosity. Analyte concentrations decreased steadily over the washout period but did not correlate across matrices for all target analytes. The study reiterates the challenges associated with determining in-competition use by relying on urinary concentrations.

Influence of multiple human chorionic gonadotropin administrations on serum and urinary steroid Athlete Biological Passport profiles in males.

The Athlete Biological Passport (ABP) is a longitudinal tool used in anti-doping to monitor biological parameters known to change with performance-enhancing drug use. The ABP consists of multiple modules, including two aimed at detecting the use of endogenous anabolic androgenic steroids: the urinary and serum steroid modules. Human chorionic gonadotropin (hCG) is a protein hormone potentially abused by male athletes to increase the production of endogenous testosterone. To date, no studies have investigated the impact of extended hCG administration on the urinary and serum steroid modules of the ABP. The goal of this study was to identify the impact of multiple hCG administrations on the parameters tracked as part of the urinary and serum steroid modules of the ABP. Ten recreationally active, healthy male individuals self-administered seven 250 µg hCG injections over 3 weeks. Serum and urine samples were collected before, during, and 2 weeks following the final injection. All ABP parameters were quantified in the respective matrix, and steroid profiles were created with Anti-Doping Administration and Management System adaptive model upper and lower limits for both matrices. In both serum and urine profiles, testosterone increased; however, the testosterone/epitestosterone ratio in urine and the testosterone/androstenedione ratio in serum showed minimal changes. Additionally, serum luteinizing hormone (LH) was quantified using an immunoassay, and a serum testosterone/LH ratio was generated. Serum LH values decreased during administration causing large increases in the serum T/LH ratio, indicating this ratio may be a more sensitive parameter for detecting hCG abuse than urinary testosterone/epitestosterone or serum testosterone/androstenedione.

Feasibility of microvolumetric capillary whole blood collections for usage in Athlete Biological Passport analysis.

The hematological module of the Athlete Biological Passport (ABP) represents an important tool in the pursuit to detect blood doping in athletes. Currently, collecting blood samples for ABP analysis can be cumbersome, invasive, and expensive, involving a venous blood draw performed by a trained phlebotomist followed by cold-chain monitored shipping to the analysis laboratory. Developing innovative methods to collect and transport ABP blood samples while adhering to strict preanalytical and analytical requirements has the potential to greatly increase testing frequency and, consequently, the effectiveness of the ABP program globally. The focus of this study was to compare venous blood collections with capillary blood collections to determine if capillary samples could be used for ABP analysis without sacrificing the analytical integrity required for antidoping testing procedures. In this study, capillary blood was collected using the Tasso+ EDTA device (Tasso, Inc.), a novel microvolumetric device that collects liquid, whole blood from skin capillaries on the upper arm. Excellent laboratory agreement was observed between venous and capillary blood samples for the three main ABP parameters: HGB, RET%, and OFF-Score. Additionally, the stability of capillary samples after storage at 4°C, similar to what would be required during transport, was acceptable for up to 72 h following collection. Finally, we generated individual ABP profiles using the adaptive model for 10 participants and observed excellent agreement between venous and capillary profiles. These results indicate capillary blood collection is a viable alternative to venous blood collections for ABP analysis.

Rsp5 and Mdm30 reshape the mitochondrial network in response to age-induced vacuole stress.

Mitochondrial decline is a hallmark of aging, and cells are equipped with many systems to regulate mitochondrial structure and function in response to stress and metabolic alterations. Here, using budding yeast, we identify a proteolytic pathway that contributes to alterations in mitochondrial structure in aged cells through control of the mitochondrial fusion GTPase Fzo1. We show that mitochondrial fragmentation in old cells correlates with reduced abundance of Fzo1, which is triggered by functional alterations in the vacuole, a known early event in aging. Fzo1 degradation is mediated by a proteolytic cascade consisting of the E3 ubiquitin ligases SCFMdm30 and Rsp5, and the Cdc48 cofactor Doa1. Fzo1 proteolysis is activated by metabolic stress that arises from vacuole impairment, and loss of Fzo1 degradation severely impairs mitochondrial structure and function. Together, these studies identify a new mechanism for stress-responsive regulation of mitochondrial structure that is activated during cellular aging.

Nucleosomes Are Essential for Proper Regulation of a Multigated Promoter in Saccharomyces cerevisiae.

Nucleosome-depleted regions (NDRs) are present immediately adjacent to the transcription start site in most eukaryotic promoters. Here we show that NDRs in the upstream promoter region can profoundly affect gene regulation. Chromatin at the yeast HO promoter is highly repressive and numerous coactivators are required for expression. We modified the HO promoter with segments from the well-studied CLN2 NDR, creating chimeric promoters differing in nucleosome occupancy but with binding sites for the same activator, SBF. Nucleosome depletion resulted in substantial increases in both factor binding and gene expression and allowed activation from a much longer distance, probably by allowing recruited coactivators to act further downstream. Nucleosome depletion also affected sequential activation of the HO promoter; HO activation typically requires the ordered recruitment of activators first to URS1, second to the left-half of URS2 (URS2-L), and finally to the right-half of URS2 (URS2-R), with each region representing distinct gates that must be unlocked to achieve activation. The absence of nucleosomes at URS2-L resulted in promoters no longer requiring both the URS1 and URS2-L gates, as either gate alone is now sufficient to promote binding of the SBF factor to URS2-R. Furthermore, nucleosome depletion at URS2 altered the timing of HO expression and bypassed the regulation that restricts expression to mother cells. Our results reveal insight into how nucleosomes can create a requirement for ordered recruitment of factors to facilitate complex transcriptional regulation.