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The aim of the current research was to develop a simple and rapid mass spectrometry-based assay for the determination of 15 steroid hormones in human plasma in a single run, which would be suitable for a routine practice setting. For this purpose, we designed a procedure based on the 2D-liquid chromatography-tandem mass spectrometry with a minimalistic sample pre-treatment. In our arrangement, the preparation of one sample takes only 10 min and can accommodate 40 samples per hour when tested in series. The following analytical run is 18 min long for all steroid hormones. In addition, we developed an independent analytical run for estradiol, significantly increasing the assay accuracy while taking an additional 10 min to perform an analytical run of a sample. The optimized method was applied to a set of human plasma samples, including chylous. Our results indicate the linearity of the method for all steroid hormones with squared regression coefficients R2 ≥ 0.995, within-run and between-run precision (RSD < 6.4%), and an accuracy of 92.9% to 106.2%. The absolute recovery for each analyzed steroid hormone ranged between 101.6% and 116.5%. The method detection limit for 15 steroid hormones ranged between 0.008 nmol/L (2.88 pg/mL) for aldosterone and 0.873 nmol/L (0.252 ng/mL) for DHEA. For all the analytes, the lowest calibration point relative standard deviation was less than 10.8%, indicating a good precision of the assay within the lowest concentration of interest. In conclusion, in this method article, we describe a simple, sensitive, and cost-effective 2D-LC/MS/MS method suitable for the routine analysis of a complex of steroid hormones allowing high analytical specificity and sensitivity despite minimal sample processing and short throughput times.

A sensitive and accurate method for determination of 17 endogenous and exogenous steroid hormones in Antarctic krill was developed. The method utilized UHPLC-MS in electrospray ionization mode (ESI). Samples were prepared by alkaline hydrolysis; sequential vortex extraction with ethyl acetate, methanol and acetonitrile; followed by a QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) clean-up method. The system suitability tests including theoretical plate number, resolution, repeatability, tailing factor proved the system's resolution and reproducibility that can meet the requirements of sample analysis. The developed method resulted in satisfactory recoveries that varied from 75.4%-110.6% and relative standard deviations (RSDs) that ranged from 3.1%-10.5%. The ranges of the limits of detection (LODs) and the limits of quantitation (LOQs) were 2-30 ng kg-1 and 10-100 ng kg-1, respectively. 14 hormones including cortisone, aldosterone, testosterone propionate, estriol, megestrol acetate, cortisone acetate, dexamethasone, testosterone, hydroxyprogesterone, nandrolone, prednisolone, cortisol, progesterone and estradiol were found in Antarctic krill. Other 3 hormones (Diethylstilbestrol, norethisterone and androsterone) were not detected. The levels of exogenous steroid hormones were much greater than those of endogenous steroid hormones, and the levels of exogenous glucocorticoids were much greater than those of exogenous sex hormones. The changes of hormones in different sex and maturity stages were also explored. Endogenous hormones might regulate the reproductive and development of Antarctic krill. The detected exogenous hormones suggests the potential for hormonal contamination in Antarctic waters that can affect organisms even affect human beings by food chain.

The first complex organisms emerged during the Ediacaran period, around 600 million years ago. The taxonomic affiliation of many of these organisms has been difficult to discern. Fossils of Dickinsonia , bilaterally symmetrical oval organisms, have been particularly difficult to classify. Bobrovskiy et al. conducted an analysis using lipid biomarkers obtained from Dickinsonia fossils and found that the fossils contained almost exclusively cholesteroids, a marker found only in animals (see the Perspective by Summons and Erwin). Thus, Dickinsonia were basal animals. This supports the idea that the Ediacaran biota may have been a precursor to the explosion of animal forms later observed in the Cambrian, about 500 million years ago. Science , this issue p. 1246 ; see also p. 1198 Lipid biomarkers extracted from organically preserved Ediacaran macrofossils unambiguously clarify their phylogeny. The enigmatic Ediacara biota (571 million to 541 million years ago) represents the first macroscopic complex organisms in the geological record and may hold the key to our understanding of the origin of animals. Ediacaran macrofossils are as “strange as life on another planet” and have evaded taxonomic classification, with interpretations ranging from marine animals or giant single-celled protists to terrestrial lichens. Here, we show that lipid biomarkers extracted from organically preserved Ediacaran macrofossils unambiguously clarify their phylogeny. Dickinsonia and its relatives solely produced cholesteroids, a hallmark of animals. Our results make these iconic members of the Ediacara biota the oldest confirmed macroscopic animals in the rock record, indicating that the appearance of the Ediacara biota was indeed a prelude to the Cambrian explosion of animal life.

Objective Supraphysiologic doses of anabolic androgenic steroids (AAS) are widely used to improve body image and sport performance goals. These substances can easily be acquired over the internet, leading to a substantial black market. We reviewed literature that assessed the quality and quantity of AAS found on the black market. Methods We searched PubMed/Medline, Embase and Google Scholar for articles published before March 2022. Additional hand searches were conducted to obtain studies not found in the primary literature search. Studies were included if they report on qualitative and/or quantitative analytical findings of AAS from the black market. Primary outcomes were proportions of counterfeit or substandard AAS. Eligible articles were extracted; quality appraisal was done using the ToxRTool for in-vitro studies. We used random-effects models to calculate the overall mean estimates for outcomes. The review protocol has been published and registered in INPLASY. Results Overall, 19 studies, which in total comprised 5,413 anabolic samples, met the inclusion criteria, and passed the quality appraisal from two WHO world regions that reported findings, the Americas and Europe. Most studies were nonclinical laboratory studies (95%) and provided samples seized by authorities (74%). In 18 articles, proportions of counterfeit substances and in eight articles, proportions of substandard substances were presented. The overall mean estimate for counterfeit anabolic steroids found on the black market was 36% (95% CI = 29, 43). An additional 37% (95% CI = 17, 63) were of substandard quality. We also demonstrate that these drugs could contain no active ingredient, or in another amount than that labeled, a wrong active ingredient, as well as not all or more active ingredients than were labeled. High heterogeneity among all analyses and significant differences between geographical subgroups were found. Conclusion With this systematic review and meta-analysis, we demonstrate that substantial mean proportions of black-market AAS are counterfeit and of substandard quality. These products pose a considerable individual and public health threat, and the very wide range in proportions of fake black-market AAS puts the user in a situation of unpredictable uncertainty. There is a great need for future prevention and harm-reduction programs to protect users from these substances.

Steroids are used as faecal markers in environmental and in archaeological studies, because they provide insights into ancient agricultural practices and the former presence of animals. Up to now, steroid analyses could only identify and distinguish between herbivore, pig, and human faecal matter and their residues in soils and sediments. We hypothesized that a finer differentiation between faeces of different livestock animals could be achieved when the analyses of several steroids is combined (Δ5-sterols, 5α-stanols, 5β-stanols, epi-5β-stanols, stanones, and bile acids). We therefore reviewed the existing literature on various faecal steroids from livestock and humans and analysed faeces from old livestock breed (cattle, horse, donkey, sheep, goat, goose, and pig) and humans. Additionally, we performed steroid analyses on soil material of four different archaeological periods (sites located in the Lower Rhine Basin, Western Germany, dating to the Linearbandkeramik, Urnfield Period / Bronze Age, Iron Age, Roman Age) with known or supposed faecal inputs. By means of already established and newly applied steroid ratios of the analysed faeces together with results from the literature, all considered livestock faeces, except sheep and cattle, could be distinguished on the basis of their steroid signatures. Most remarkably was the identification of horse faeces (via the ratio: epi-5β-stigmastanol: 5β-stigmastanol + epicoprostanol: coprostanol; together with the presence of chenodeoxycholic acid) and a successful differentiation between goat (with chenodeoxycholic acid) and sheep/cattle faeces (without chenodeoxycholic acid). The steroid analysis of archaeological soil material confirmed the supposed faecal inputs, even if these inputs had occurred several thousand years ago.

Abstract Context Determination of steroid levels in the amniotic fluid gives some insight on fetal adrenal and gonadal functions. Objective Our objectives were to establish reference ranges of 12 steroid levels throughout pregnancy and to compare them with steroid levels from pregnancies with fetuses presenting with 21-hydroxylase deficiency (21OHD). Methods Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was applied to 145 “control” amniotic fluid samples from gynecology activity (12 + 6 to 32 + 4 gestational weeks, GW). The following steroids were analyzed according to gestational age and compared to 23 amniotic fluid samples from fetuses with classic 21OHD confirmed by molecular studies: delta-4-androstenedione (D4), dehydroepiandrosterone (DHEA), 17-hydroxyprogesterone (17OHP), 11-deoxycortisol (11OH), 21-deoxycortisol (21OH), corticosterone, deoxycorticosterone (DOC), testosterone, pregnenolone, 17-hydroxypregnenolone (17Pregn), cortisol, and cortisone. Chromosomal sex was determined by karyotype and gestational age by biometric measurements. Results Analysis of control samples showed a statistically significant difference for D4 and testosterone levels according to fetal sex. Cortisol, corticosterone, and DOC had lower concentrations before 20 GW than after 20 GW, whereas 17Pregn and pregnenolone had higher concentrations before 20 GW. This allowed us to establish age- and sex-dependent reference values. We observed higher 21OH, 17Pregn, D4, and testosterone levels in females with 21OHD than female controls. The ratios 17OHP/17Pregn, D4/DHEA, and 11OH/17OHP appeared discriminant for the diagnosis of 21OHD. Conclusion Our study provides information on fetal steroidogenesis and suggests reference values for 12 steroids during pregnancy. This allows a prenatal diagnosis of 21OHD within 24 hours and might be useful in the diagnosis of other variations of sex development.

Steroid hormones and their respective nuclear receptors are essential mediators in numerous physiologic and pathophysiologic processes, ranging from regulation of metabolism, immune function, and reproductive processes to the development of hormone-dependent cancers such as those of the breast and prostate. Because steroids must enter cells before activating nuclear receptors, understanding the mechanisms by which cellular uptake occurs is critical, yet a clear understanding of these mechanisms has been elusive. It is generally assumed that diffusion-driven uptake is similar across various steroids whereas an elevated cellular concentration is thought to reflect active uptake, but these assumptions have not been directly tested. Here we show that intact cells rapidly accumulate free steroids to markedly elevated concentrations. This effect varies widely depending on steroid structure; more lipophilic steroids reach more elevated concentrations. Strong preferences exist for 3β-OH, Δ5-steroids vs. 3-keto, Δ4-structural features and for progestogens vs. androgens. Surprisingly, steroid-structure-specific preferences do not require cell viability, implying a passive mechanism, and occur across cells derived from multiple tissue types. Physiologic relevance is suggested by structure-specific preferences in human prostate tissue compared with serum. On the other hand, the presence of serum proteins in vitro blocks much, but not all, of the passive accumulation, while still permitting a substantial amount of active accumulation for certain steroids. Our findings suggest that both passive and active uptake mechanisms make important contributions to the cellular steroid uptake process. The role of passive, lipophilicity-driven accumulation has previously been largely unappreciated, and its existence provides important context to studies on steroid transport and action both in vitro and in vivo.

Molecular biomarkers preserved in lake sediments are increasingly used to develop records of past organism occurrence. When linked with traditional paleoecological methods, analysis of molecular biomarkers can yield new insights into the roles of herbivores and other animals in long-term ecosystem dynamics. We sought to determine whether fecal steroids in lake sediments could be used to reconstruct past ungulate use and dominant taxa in a small catchment in northern Yellowstone National Park. To do so, we characterized the fecal steroid profiles of a selection of North American ungulates historically present in the Yellowstone region (bison, elk, moose, mule deer, and pronghorn) and compared them with those of sediments from a small lake in the Yellowstone Northern Range. Analysis of a set of fecal steroids from herbivore dung (Δ 5 -sterols, 5α-stanols, 5β-stanols, epi5β-stanols, stanones, and bile acids) differentiated moose, pronghorn, and mule deer, whereas bison and elk were partially differentiated. Our results show that bison and/or elk were the primary ungulates in the watershed over the past c . 2300 years. Fecal steroid influxes reached historically unprecedented levels during the early and middle 20 th century, possibly indicating high local use by ungulates. Comparison of fecal steroid influxes with pollen and diatom data suggests that elevated ungulate presence may have contributed to decreased forage taxa (Poaceae, Artemisia , and Salix ), relative to long-term averages, and possibly increased lake production. Our results reflect past change within a single watershed, and extending this approach to a network of sites could provide much-needed information on past herbivore communities, use, and environmental influences in Yellowstone National Park and elsewhere.

Population-based assays have been employed extensively to investigate the interactions of transcription factors (TFs) with chromatin and are often interpreted in terms of static and sequential binding. However, fluorescence microscopy techniques reveal a more dynamic binding behaviour of TFs in live cells. Here we analyse the strengths and limitations of in vivo single-molecule tracking and performed a comprehensive analysis on the intranuclear dwell times of four steroid receptors and a number of known cofactors. While the absolute residence times estimates can depend on imaging acquisition parameters due to sampling bias, our results indicate that only a small proportion of factors are specifically bound to chromatin at any given time. Interestingly, the glucocorticoid receptor and its cofactors affect each other’s dwell times in an asymmetric manner. Overall, our data indicate transient rather than stable TF-cofactors chromatin interactions at response elements at the single-molecule level. Transcription factors (TFs) are thought to regulate gene expression by stably binding to target DNA elements. Here, the authors use single-molecule tracking to analyse the dynamic behaviour of steroid receptors TFs and show that most specific interactions with chromatin are transient and dynamic.

The addition of palbociclib to fulvestrant prolonged overall survival among women with hormone receptor–positive, HER2-negative advanced breast cancer who had sensitivity to previous hormonal therapy. In the entire trial group, survival differences were not significant.