Analysis Of Steroids Research Articles

Rapid Quantification of Endogenous Steroids in Human Serum Using Leidenfrost Effect-Assisted Thermal Desorption Atmospheric Pressure Photoionization Orbitrap Mass Spectrometry.

Unconjugated sex steroids in human serum play a crucial role in physiological and pathological studies and are frequently considered as biomarkers in clinical diagnosis. Because of their low polarity, poor volatility, and low concentration, the rapid and highly sensitive analysis of sex steroids in real serum matrix by ambient mass spectrometry is still challenging. Here, Leidenfrost effect-assisted thermal desorption atmospheric pressure photoionization orbitrap mass spectrometry (LETD-APPI-MS) was developed and applied to quantify free sex steroids in human serum without derivatization and chromatography separation within a few minutes. The concentration of target analyte could be increased by approximately two orders during the LETD process. The limit of quantifications and detections of endogenous sex steroids in human serum were measured at the ppt level. In contrast with commonly used immunoassays in clinical laboratories, LETD-APPI-MS enables the accurate measurements of multiple free sex steroids without the interference of cross-reactions. The endogenous sex steroids of 38 female serums at four physiological stages during pregnancy were rapidly tested by LETD-APPI-MS, whose results were highly consistent with that using liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC-APCI-MS), indicating LETD-APPI-MS has a strong clinical application potential in steroid analysis.

Recurrent Hypokalemia and Adrenal Steroids in Patients With APECED.

ContextHypokalemia is a common finding in patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) but its exact cause often remains unknown.ObjectiveTo explore the prevalence and etiology of hypokalemia and the role of adrenal steroids therein in a cohort of patients with APECED.MethodsWe performed a cross-sectional assessment and retrospective data collection on 44 Finnish patients with APECED to identify subjects with episodes of hypokalemia. Also 68 healthy matched controls attended the cross-sectional evaluation. Factors associating with a tendency for hypokalemia were analyzed by reviewing medical records during 1960-2021 and performing a cross-sectional analysis of serum adrenal steroids.ResultsIn total 14 of the 44 APECED patients (32%) had episodes of hypokalemia; 2 presented with hypokalemia at cross-sectional evaluation and 12 had a history of hypokalemia before the cross-sectional evaluation. Hypokalemic episodes started at the median age of 14.1 years; 12/14 (86%) had primary adrenal insufficiency (PAI). The median number of hypokalemic periods per year was 0.3 (range 0.04-2.2); the frequency correlated positively with the number of clinical APECED manifestations at the time of cross-sectional evaluation (r=0.811, p<0.001). Etiologies of hypokalemia varied but episodes often occurred when new clinical manifestations developed and during hospitalizations. Three patients had kidney defects, also associated with electrolyte imbalances. Severity of hypokalemia varied (range 2.2-3.2 mmol/L), but no severe complications were observed. At cross-sectional evaluation, patients with PAI (n = 30) had significantly lower median plasma potassium and higher sodium concentration than controls, suggesting that fludrocortisone treatment contributed to hypokalemia. Detailed analysis of adrenal steroids provided no conclusive differences between patients with and without episodes of hypokalemia.ConclusionsIn APECED, hypokalemia is common and varies in terms of frequency, etiology, and severity. PAI and kidney disease predispose to hypokalemia. In addition, hypokalemic periods seem to be more common in patients with more severe phenotype of APECED.

Development of a Liquid Chromatography-Mass Spectrometry-Based In Vitro Assay to Assess Changes in Steroid Hormones Due to Exposure to Per- and Polyfluoroalkyl Substances.

Per- and poly-fluorinated substances (PFASs) are organic pollutants that have been linked to numerous health effects, including diabetes, cancers, and dysregulation of the endocrine system. This study aims to develop a liquid chromatography with tandem mass spectrometry (LC-MS/MS) assay to measure changes in 17 hormones in H295R cell line (a steroid producing adrenocortical cells) upon exposure to PFASs. Due to the challenges in the analysis of steroid hormones using electrospray ionization MS, a chemical derivatization method was employed to achieve 0.07-2 μg/L detection limits in LC-MS/MS. Furthermore, a 10-fold concentration factor through solid-phase extraction (SPE) allows for consistent sub-parts per billion detections. Optimization of the derivatization conditions showed doubly-derivatized products in some hormone analytes, including progesterone, corticosterone, and cortisol, and gave improved ionization efficiency up to 20-fold higher signal than the singly-derivatized product. The use of SPE for sample cleanup to analyze hormones from cellular media using weak anion exchange sorbent yielded 80-100% recovery for the 17 targeted hormones. The method was validated by exposing H295R cells to two known endocrine disruptors, forskolin and prochloraz, which showed expected changes in hormones. An initial exposure of H295R cells with various PFAS standards and their mixtures at 1 μM showed significant increases in progestogens with some PFAS treatments, which include PFBS, PFHxA, PFOS, PFDA, and PFDS. In addition, modest changes in hormone levels were observed in cells treated with other sulfonated or carboxylated headgroup PFASs. This sensitive LC-MS/MS method for hormone analysis in H295R cells will allow for the investigations of the alterations in the hormone production caused by exposure to various environmental insults in cell-based assays and other in vitro models.

Improvement of soil fertility in historical ridge and furrow cultivation

AbstractRidge and furrow cultivation is the most widely used agricultural technique in medieval and postmedieval Europe, but the fertilization of soils during their use is not yet fully understood. Pedological analyses of this cultivation technique provided information, which led to the assumption that some of the investigated sites in Northern and Central Germany were manured with livestock excrements during cultivation. The objective of this study is to determine whether and how the soils have been fertilized and which materials were applied for this purpose. We investigated soils at five sites using phosphate and steroid analyses (stanols and bile acids), black carbon analyses, and a micromorphology study. The results showed that livestock waste was likely used as fertilizer at four of the five studied sites at low intensities, with pigs and herbivores being the probable sources of the excrement. But also the application of human feces to the soil might be possible at least at one site. Often used agricultural methods such as plaggen cultivation and an intentional charcoal input to enhance soil fertility could not be clearly verified for our study sites.

Analysis of fatty acids and sex steroid hormones in rainbow trout testes ( Oncorhynchus mykiss ) during the reproductive process

In the present study, we aimed to examine the cellular structure of the testes, the hormonal changes occurring throughout the reproductive cycle, and the fatty acid composition in rainbow trout. An electron microscopic analysis was conducted to investigate the morphology and cellular changes during the development of the testes. Germ cells were observed in different stages of differentiation: primary spermatogonia (PSGs), secondary spermatogonia (SSGs), primary spermatocytes (PSCs), secondary spermatocytes (SSCs), spermatids (SPDs) and spermatozoa (SZs). Differentiated spermatozoa were observed within the cysts. Regarding steroid hormones, the maximum level of testosterone was in the mature stage, and the minimum level was in the spent stage; for 11-ketotestosterone, the highest level was in the maturing stage and the lowest level was in the spent stage. In terms of the fatty acid composition, the levels of C14, C15, C16, C18, C16:1n7, C18:1n7, C18:1n9, C20:4n6, n-3 PUFAs, n-6 PUFAs, C20:5n-3 (EPA), C22:5n-3 (DPA) and C22:6n3 (DHA) (g−1 of gonad wet weight) were the lowest during the immature stage and the highest during the mature stage of the testes. The major significance of the study is the sex steroid hormonal profile, the primary regulators of spermatogenesis and the fatty acid composition, which is important for the development of gonads and sperm. These data should contribute to the knowledge regarding the reproductive biology of male rainbow trout, for which research data remain scarce.

Evaluating suitability of saliva to measure steroid concentrations in grey seal pups

Measurement of steroids in wild pinnipeds can facilitate assessment of breeding, nutritional and stress status, and is useful in understanding behavioral responses. Even in young animals, sex steroids may be important in behavioral interactions and immune modulation. Use of saliva can avoid the large fluctuations seen in some steroids in plasma, and can negate the need for venipuncture, making it a potentially useful matrix in the wildlife. However, its utility in estimating steroid levels in wild young pinnipeds has not been evaluated. Here, we investigated the suitability of saliva for steroid hormone analysis in wild grey seal pups during their suckling and post-weaning fast periods. We collected saliva (n = 38) and plasma (n = 71) samples during the breeding season on the Isle of May, Scotland, 2012. We investigated success of sample collection, ease of preparation, accuracy and precision of analysis, and, where possible, comparability of measurements (n = 27) from saliva and plasma. Plasma sampling was rapid, whereas sampling saliva took up to five times longer. Analytical performance criteria (parallelism, accuracy, and precision (intra and inter assay co-efficient of variation (% CV)) of commercial ELISA kits to measure estradiol, testosterone and cortisol in both matrices were assessed. Estradiol and cortisol assays performed well and can be used in plasma and saliva. However, we could not confidently validate testosterone for either matrix. Saliva estradiol correlated with levels in plasma. Saliva sample preparation was faster and simpler than plasma preparation because it did not require extraction. However, given the additional time taken to obtain saliva in the wild, the possibility of blood contamination from oral damage and the lower success rate in obtaining sufficient sample for analysis, we recommend that this matrix only be used as an alternative to plasma sampling measurement in pinnipeds when animals are anaesthetized, tolerate mouth swabbing, or have been trained to accept saliva sampling in captivity.

Biochemical Analyses of Ten Cyanobacterial and Microalgal Strains Isolated from Egyptian Habitats, and Screening for Their Potential against Some Selected Phytopathogenic Fungal Strains

Microalgae and cyanobacteria are rich sources of numerous phytochemical compounds with intrinsic antifungal potential. This research aimed to screen the phytochemical compounds and contents, as well as the antioxidant profiles, in eight cyanobacterial and two microalgal strains isolated from soil and brackish water habitats in Egypt. Our study also evaluated their antifungal activities against three phytopathogenic fungi—namely, Pythium ultimum, Fusarium solani, and Botryodiplodia theobromae, which are known to cause severe plant loss. The biochemical compounds were obtained from the cyanobacterial and algal methanolic extracts, and were identified through comparative phytochemical analyses related to the inhibition of the fungal pathogens. Comparative qualitative analyses of alkaloids, steroids, glycosides, and saponins were also carried out. The quantitative phytochemical screening of the cyanobacterial and algal strains investigated revealed the presence of xylanase, glucanase, and chitinase enzymes, along with some bioactive compounds, such as phenolics, flavonoids, proteins, neutral sugars, and carotenoids, which were species-dependent and detected in variable amounts in the extracts. The unicellular green microalgal strain Dunaliella sp. HSSASE13 displayed the highest level of antioxidant activity. However, the highest antifungal activities were shown by the heterocystous cyanobacterial strain Anabaena sp. HSSASE11 (83.94%), followed by Dunaliella sp. HSSASE13 (81.94%) and the non-heterocystous cyanobacterial strain Oscillatoria nigro-viridis HSSASE 15 (63.42%), against the three fungal pathogens B. theobromae, F. solani, and P. ultimum, respectively. Our results indicate that the highest significant and positive correlations of flavonoids (r = 0.854), phenolics (r = 0.785), DPPH scavenging activity (r = 0.876), total proteins (r = 0.808), xylanase activity (r = 0.876), glucanase activity (r = 0.746), and total neutral sugars (r = 0.764), in terms of their antifungal activities, were recorded against F. solani. Conclusively, the cyanobacterial and algal strains tested in the present study can be useful agents for the management and biocontrol of plant-infecting fungal pathogens.

Validation of steroid ratios for random urine by mass spectrometry to detect 5α-reductase deficiency in Vietnamese children.

The 5α-reductase-type-2 deficiency (5ARD2) is a rare autosomal recessive 46,XY disorder of sex development caused by the mutated 5α-reductase type 2 (SRD5A2) gene. In this disease, defective conversion of testosterone to dihydrotestosterone leads to variable presentations of male ambiguous genitalia during fetal development. We aimed to examine characteristics of patients presenting with 5ARD2 over a 4year period. Random urine samples of control and patients with suspected 5ARD2 were collected and urine steroidomic metabolites were measured by Gas chromatography-mass spectrometry (GC-MS) in the period from 2017 to 2021 at National Children's Hospital, Hanoi Vietnam. 5α- to 5β-reduced steroid metabolite ratio, 5a-tetrahydrocortisol to tetrahydrocortisol (5α-THF/THF), was reviewed by receive operator characteristics (ROC) curve analysis. Molecular testing was offered to 25 patients who were diagnosed with 5ARD2 by GC-MS urinary steroid analysis. Urine steroidomic profiling was conducted for 104 male controls and 25 patients between the ages of 6months and 13years old. Twelve of the twenty-five 5ARD2 patients agreed to undertake genetic analysis, and two mutations of the SRD5A2 gene were detected in each patient, confirming the diagnosis. All patients showed a characteristically low ratio of 5α-THF/THF. There was no overlap of 5α-THF/THF ratio values between control and 5ARD2 groups. The ROC of 5α-THF/THF ratio at 0.19 showed 100% sensitivity and 100% specificity for boys between 6months and 13years of age. Analysis of the urine steroid metabolome by GC-MS can be used to assist in the diagnosis of 5ARD2. We recommend consideration of random urine steroid analysis as a first-line test in the diagnosis of 5ARD2.

Effect of temperature on sex and steroid hormones of purple mud crab, Scylla tranquebarica (Fabricius, 1798) during egg incubation, larvae rearing and juvenile production

The influence of temperature on sex determination has been reported previously on many crustaceans. The interpretability of temperature capability to regulate the steroidogenic pathway, which in turn governs the offspring sex of mud crab, still remains unclear. Thus, the aim was to determine the effects of temperature on sex percentage and steroidal pathway in purple mud crab, Scylla tranquebarica. In sex percentage analysis, the eggs (28°C and 30°C), larvae (28°C, 30°C and 32°C) and juveniles (26°C, 28°C, 30°C) were exposed to different temperatures. The results revealed that exposure to a higher temperature during larvae (32°C) and juvenile (30°C) stages probably initiated the maleness (58.7%) and femaleness (60.8%) mechanism respectively. In steroid hormone analysis, the eggs (26°C, 28°C, 30°C and 32°C), larvae (29°C, 30°C, 31°C and 32°C) and juveniles (26°C, 28°C, 30°C and 32°C) were exposed to different temperatures. The steroidal assay corroborated the results of the sex percentages, which found higher dihydrotestosterone (172.4 pg/g) and testosterone (580.2 pg/g) levels when the larvae were incubated at a higher temperature (32°C) while higher estrone (136.9 pg/g) and estradiol (540.7 pg/g) levels when the juveniles were reared at high temperature (30°C). In brief, the temperature plays a significant role to redirect the mud crab sex during the larval and juvenile stages.

An LC-MS/MS method for the simultaneous quantification of 32 steroids in human plasma

A development of robust and rapid method with simple sample preparation for the analysis of steroids of C18-, C19-, C21- families is of interest of many research groups. Here we present a novel LC-MS/MS method for the simultaneous quantification of 32 steroid hormones in human plasma. Twenty-two of them were analyzed directly without the need for derivatization, while ten were derivatized with 2-fluoro-1-methylpyridinium p-toluenesulfonate. The steroids were separated on a C18 column with a gradient elution consisting of methanol and water with the addition of 0.1% formic acid. The mass spectrometer was operated in positive ESI mode. Validation demonstrated that the method was applicable for the quantitative analysis of two C18- steroids (estrone, estradiol), nineteen C19- steroids (testosterone, epitestosterone, dihydrotestosterone, 11-ketodihydrotestosterone, 11β-hydroxyandrostenedione, 11β-hydroxytestosterone, 11-ketotestosterone, dehydroepiandrosterone, 7α-hydroxydehydroepiandrosterone, 7β-hydroxydehydroepiandrosterone, 7-ketodehydroepiandrosterone, androsterone, epiandrosterone, androstenedione, androstenediol, 5α-androstane-3α,17β-diol, 5α-androstane-3β,17β-diol, 5β-androstane-3α,17β-diol, 5β-androstane-3β,17β-diol), and eleven C21- steroids (cortisol, 21-deoxycortisol, 11-deoxycortisol, cortisone, corticosterone, 11-deoxycorticosterone, pregnenolone, 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone, 5α-dihydroprogesterone). The lower limits of quantification are appropriate for analyses in both physiological and various pathophysiological conditions. The accuracy, intra- and inter-day precision values as well as stability tests were in accordance with FDA Guidelines. The method will be a useful tool in investigating the mechanisms of steroid-related diseases and will serve as a steppingstone for the development of other methods for steroid analyses in various biological matrices such as prostate tissue, cerebrospinal fluid, urine, seminal fluid, and saliva.

Assessment of automated off-line solid-phase extraction LC-MS/MS to monitor EPA priority endocrine disruptors in tap water, surface water, and wastewater.

EPA method 539.1 recently introduced an expanded list of priority endocrine-disrupting compounds (EDCs), some of which were also included in the Unregulated Contaminant Monitoring Rule 3 (UCMR3). Though standardized methods are available for drinking water, analysis of steroid hormones and bisphenol A (BPA) at the ultra-trace level remains challenging. This study set out to evaluate the suitability of automated off-line solid-phase extraction (SPE) liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) for the determination of EPA-priority EDCs in environmental water matrixes (tap water, surface water, and wastewater influents and effluents). The target molecules included 14 steroid hormones (altrenogest, androstenedione, equilenin, equilin, α-estradiol, β-estradiol, estriol, estrone, ethinylestradiol, levonorgestrel, medroxyprogesterone, norethindrone, progesterone, testosterone) and BPA. Factors that may influence the analytical performance were assessed. This involved, for instance, testing combinations of SPE materials from different brands and protocol variations. Several materials presented absolute extraction efficiencies in acceptable ranges. Initial sample pH, nature of reconstitution medium, and mobile phase salt concentration were among the potential factors affecting analyte signal. Storage conditions (different preservative agents) possibly exerted the strongest influence, in agreement with the literature. Limits of detection were in the range of 0.03-0.5ng/L in drinking water, 0.1-0.5ng/L in surface water, and 0.16-1ng/L in wastewater. Method validation also involved testing linearity, accuracy, and precision in reagent water and matrix-matched extracted calibrants. The method was applied to field-collected water samples in Eastern Canada. Summed EDC concentrations remained low in tap water (<LOQ-0.92ng/L), while higher detection frequencies and contamination levels were reported in riverine surface waters (2.6-37ng/L) and municipal wastewaters (10-424ng/L).

Imaging of Corticosteroids in Mouse Kidney Using Mass Spectrometry

Aldosterone regulates renal sodium reabsorption which is important for blood pressure homeostasis. Glucocorticoids may also contribute to this process although this is less well understood. Analysis of steroid hormones and the metabolites in the plasma and urine gives insights into corticosteroid biology but steroid concentrations at a cellular level are largely unknown, and the kidney remains a “black box”. Mass spectrometry imaging (MSI) permits localisation of steroids in histological zones based on regional markers. This approach has been previously applied to localise steroids in brain and testes, and here is applied to kidney. Our aim was to map and quantify glucocorticoids and aldosterone in different histological zones (cortex, medulla) of kidneys from mice receiving different dietary salt, to provide new information relevant to the physiological actions of these hormones.Cryosections kidney from male C57BL6 mouse (age 12 weeks, n=6: dietary salt low 0.03% vs normal 0.3% vs high 3%) were subject to MSI analysis following Girard T reagent derivatisation and α‐cyano‐4‐hydroxycinnamic acid matrix application. Matrix assisted laser desorption/ionisation (MALDI) was used as a sampling method, coupled to Fourier Transform Ion cyclotron mass spectrometry.Ions with m/z 458.3010 (Δppm=0.65), 460.3166 (Δppm=0.65), and 474.2957 (Δppm=1.05) were detected, using MALDI, in renal sections for derivatives of 11‐dehydrocorticosterone, corticosterone and aldosterone respectively. Untargeted evaluation of ions was conducted to find regional markers that would allow definition of kidney histological zones. Heat maps indicated that corticosterone intensity was higher in the inner cortex than the rest of the kidney. In contrast 11‐dehydrocorticosterone, a metabolite produced by the enzymic activity of 11β‐hydroxysteroid dehydrogenase type II, was detected at highest levels in the medulla. The aldosterone signal was equally strong in medulla and outer cortex. Steroid localisation after treatment with different salt intakes indicated that the intensity of the corticosterone signal increases in the outer cortex in kidneys from mice receiving low salt diet, while 11‐dehydrocorticosterone was not affected. The intensity of the aldosterone signal within the kidney was not changed by different salt intakes. Our approach shows that it is feasible to image corticosteroids at 75μm spatial resolution within the kidney. This technology provides new insights into the physiological control of sodium transport by steroids and opens doors to understanding changes in disorders of blood pressure.

Metabolic Alteration Analysis of Steroid Hormones in Niemann-Pick Disease Type C Model Cell Using Liquid Chromatography/Tandem Mass Spectrometry.

Niemann–Pick disease type C (NPC) is an autosomal recessive disease caused by a functional deficiency of cholesterol-transporting proteins in lysosomes, and exhibits various clinical symptoms. Since mitochondrial dysfunction in NPC has recently been reported, cholesterol catabolism to steroid hormones may consequently be impaired. In this study, we developed a comprehensive steroid hormone analysis method using liquid chromatography/tandem mass spectrometry (LC–MS/MS) and applied it to analyze changes in steroid hormone concentrations in NPC model cells. We investigated the analytical conditions for simultaneous LC–MS/MS analysis, which could be readily separated from each other and showed good reproducibility. The NPC phenotype was verified as an NPC model with mitochondrial abnormalities using filipin staining and organelle morphology observations. Steroid hormones in the cell suspension and cell culture medium were also analyzed. Steroid hormone analysis indicated that the levels of six steroid hormones were significantly decreased in the NPC model cell and culture medium compared to those in the wild-type cell and culture medium. These results indicate that some steroid hormones change during NPC pathophysiology and this change is accompanied by mitochondrial abnormalities.

A quantitative ultra-performance liquid chromatography high-resolution mass spectrometry analysis of steroids from human scalp hair

Hair steroid analysis offers the possibility to evaluate long-term steroid metabolism. It is especially beneficial when studying the steroid milieu in newborns and children, for example it can provide new insights into steroid metabolism over months and is unaffected by diurnal fluctuations in steroid concentrations. This study describes a quantitative and highly selective high-resolution mass spectrometry method for the simultaneous analysis of multiple steroids from human scalp hair. The developed method utilizes parallel reaction monitoring in the analysis of hydroxylamine derivatized steroids from hair samples first washed with isopropanol, extracted with methanol, and further purified with solid-phase extraction and liquid-liquid extraction. The method was proven suitable for the quantitative analysis of endogenous glucocorticoids (cortisol [1.5–364 pg/mg]; cortisone [3.6–355 pg/mg]; corticosterone [0.7–347 pg/mg]; 11-deoxycortisol [0.1–343 pg/mg]), androgens (testosterone [0.1–288 pg/mg]; dehydroepiandrosterone [0.3–288 pg/mg]; androstenedione [0.1–285 pg/mg]; 11β-hydroxyandrostenedione [0.3–304 pg/mg]), progestogens (pregnenolone [0.1–313 pg/mg]; progesterone [0.1–313 pg/mg]; 17α-hydroxypregnenolone [0.3–315 pg/mg]; 17α-hydroxyprogesterone [0.7–330 pg/mg]; 21-hydroxyprogesterone [0.6–320 pg/mg]), and estrogens (estrone [0.1–267 pg/mg]; estradiol [0.5–274 pg/mg]). In addition, 11-ketoandrostenedione [0.6–60 pg/mg]; dihydrotestosterone [0.3–290 pg/mg]; 11-ketotestosterone [0.1–12 pg/mg]; and 5α-androstanedione [0.6–281 pg/mg] could be analyzed semi-quantitatively. Aldosterone [3.5–346 pg/mg]; 11β-hydroxytestosterone [0.3–300 pg/mg]; and 11-ketodihydrotestosterone [0.3–300 pg/mg] were also included in the method, but their concentrations were below the lower limit of quantification in all tested hair samples. The method was applied for the analysis of authentic hair samples from different age groups ranging from newborns to adults, including mothers within 48 h after delivery. The newborn hair samples displayed the widest variety and had also the highest amounts of steroids in comparison to the samples of the other groups.

Comparison of steroid and thyroid hormone concentrations in blood serum and plasma of captive tigers.

Analysis of steroid and thyroid hormones is often performed in blood serum. Occasionally though, plasma samples are submitted in lieu of serum for exotic species such as tigers. However, blood tube anticoagulants may affect hormone values. We compared serum and heparin plasma results for 7 hormones in tigers. Serum and plasma samples were collected from 25 tigers and analyzed for progesterone, 17-hydroxyprogesterone, cortisol, androstenedione, testosterone, estradiol, and thyroxine. Using Lin concordance correlation, serum and heparin plasma measures agreed for all hormones except cortisol. However, Passing-Bablok regression only found agreement between serum and heparin plasma measures for androstenedione, testosterone, and estradiol. Median values between the 2 sample types were significantly (p < 0.05) different for progesterone, 17-hydroxyprogesterone, cortisol, and thyroxine. Our results suggest that, for the aforementioned hormones, serum and heparin plasma values may not always be comparable.

3D printing of compact electrochemical cell for sequential analysis of steroid hormones

3D printing technology revolutionized the development of electrochemical sensors allowing the fabrication of custom devices that can be adapted to meet specific applications. This study describes the development of a miniaturized electrochemical cell containing integrated electrodes manufactured entirely by 3D printing for the sequential detection of steroid hormones. The electrochemical sensor was manufactured in a single step by combining two types of thermoplastic filaments. Polylactic acid-Carbon Black (PLA-CB) was used in the electrode region and acrylonitrile butadiene styrene (ABS) was used in the insulating region. Chemical/electrochemical treatments were used to remove the inert polymer matrix and expose the electroactive sites of the CB electrodes. These pre-treatment procedures significantly enhanced the electrochemical response of the printed sensor. The analytical performance of the activated 3D printed electrochemical cell was demonstrated in the sequential detection of estradiol (E2) and progesterone (P4) in artificial urine. The electrochemical cell offered good repeatability (RSD = 3.1%) and reproducibility (RSD = 10.7%) between analyses reaching detection limits of 0.11 and 17.8 µmol L-1 for E2 and P4, respectively. In this sense, these results demonstrate that the use of 3D printing paves the way for the fast and economical fabrication of miniaturized electrochemical cells for hormone detection in biological fluids.

Rapid Identification of 44 Steroids in Human Urine Samples using HPLCESI- QTOF-MS

Objective: Detailed analysis of un-processed and un-derivatized free and conjugated urinary steroids is useful to avoid miscalculations and to diagnose sports doping and adrenal problems, including abnormal steroidogenesis, congenital deficiency of related enzymes, cancer, and other disease conditions. Hence, the present study was conducted to develop a soft ionization method to identify the maximum number of urinary steroids using ultra-performance liquid chromatography coupled with quadrupole time of flight mass spectrometer (HPLC–Q-TOF-MS). Material and Methods: HPLC–Q-TOF-MS was carried out for the qualitative detection of steroids and their conjugates in urine samples. The method provides high sensitivity and fast analysis of steroids and their glucuronides without hydrolysis or sample preparation or extraction of steroids. Results: Using the method, 44 steroids belonging to C-18, C-19, and C-21 classes and their conjugates were resolved and identified using positive and negative modes of ionizations by their characteristic ionization and collision energy induced dissociation behaviors. Conclusion: The method is time-saving and good to compare samples from different peoples with control or healthy ones as it does not require any kind of pre-treatment or sample processing. It provides a complete picture of steroids metabolism and catabolism. It can be good for doping control or to explore the effects of other drugs. However, in qualitative analysis, one may miss the significant information unless direct methods of steroids analysis to be employed.

An LC-MS/MS Methodological Framework for Steroid Hormone Measurement from Human Serum.

Estrogens and androgens are important regulators of sexual development and physiological processes in men and women, acting on numerous organs throughout the body. Moreover, they can contribute to a variety of pathologies, including osteoporosis, cancer, and cardiovascular and neurologic diseases. Analysis of estrogens and androgens in biological samples has been commonly performed using immunoassays for many years. However, these assays are suboptimal, as there is cross-reactivity with similar analytes, and they have moderate specificity and sensitivity. Thus, there is a clinical need to develop highly sensitive and specific methods for the accurate measurement of estrogen and androgen concentrations. Herein, we describe the development of three liquid chromatography coupled tandem mass spectrometry-based methods that incorporate the use of a Triple Quadrupole Mass Spectrometer for quantitative measurement of endogenous concentrations of various steroid hormones in human serum samples: (1) the simultaneous measurement of testosterone, androstenedione, and cortisol, (2) dehydroepiandrosterone (DHEA), and (3) 17β-estradiol (E2). The use of derivatizing reagents, Girard's reagent P and dansyl chloride, allowed for significant gains in sensitivity in the analysis of DHEA and E2, respectively, relative to the underivatized analyte. These procedures proved efficient and adequately sensitive for steroid hormone analysis in extracted patient sera samples from older men and postmenopausal women, providing reliable data down to low nanogram/ml and sub-nanogram/ml levels. Moreover, utilizing the combination of highly specific mass transitions associated with these analytes and their respective internal deuterated standards provided a high degree of specificity to the identity of these hormones.

Connecting the spots: Understanding cheetah reproduction to improve assisted breeding and population management

The North American cheetah (Acinonyx jubatus) population serves as both an insurance population for their rapidly decreasing wild cohorts as well as a research population to understand the unique biology of this species. This review focus on the complexity of the female cheetah reproductive system and the recent advances that have been made towards understanding basic biology and reproductive function, and application of assisted breeding technologies to enhance reproduction and maintain genetic diversity of this species in human care. Cheetah females are non-seasonal breeders that exhibit lengthy periods of anestrus that are not associated with age, environment, or reproductive potential. It is possible to collect good quality oocytes, that support fertilisation and successful early embryonic development, regardless of female age (from 2 to 12 yr old). However, the prevalence of uterine pathologies increases with age and prevents middle to advanced age females from establishing pregnancy. Pregnancy can be diagnosed in non-sedated cheetah females via ultrasonography (first month), steroid hormone analysis (second/third month) or radiography (third month). Fecal biomarkers, such as Immunoglobulin J, show great promise for diagnosing pregnancy at an early stage as well as other physiological states. Several decades of basic research have led to efficient management of natural breeding and recent successes in assisted reproduction.

Human and livestock faecal biomarkers at the prehistorical encampment site of Ullafelsen in the Fotsch Valley, Stubai Alps, Austria – potential and limitations

Abstract. The Ullafelsen at 1869 m above sea level (a.s.l.) in the Tyrolean Stubai Alps next to Innsbruck is an important (geo)archeological reference site for the Mesolithic period. Buried fireplaces on the Ullafelsen plateau were dated at 10.9 to 9.5 ka cal BP and demonstrate together with thousands of flint stone artifacts the presence of hunter-gatherers during the Early Holocene. Grazing livestock has been a predominant anthropozoological impact in the Fotsch Valley presumably since the Bronze Age (4.2–2.8 ka). In order to study the human and/or livestock faeces input on the Ullafelsen, we carried out steroid analyses on 2 modern ruminant faeces samples from cattle and sheep, 37 soil samples from seven archeological soil profiles, and 9 soil samples from five non-archeological soil profiles from the Fotsch Valley used as reference sites. The dominance of 5β-stigmastanol and deoxycholic acid in modern cattle and sheep faeces can be used as markers for the input of ruminant faeces in soils. The OAh horizons, which have accumulated and developed since the Mesolithic, revealed high contents of steroids (sterols, stanols, stanones and bile acids); the eluvial light layer (E (LL)) horizon coinciding with the Mesolithic living floor is characterized by medium contents of steroids. By contrast, the subsoil horizons Bh, Bs and BvCv contain low contents of faecal biomarkers, indicating that leaching of steroids into the podsolic subsoils is not an important factor. High content of 5β-stigmastanol and deoxycholic acid in all soil samples gives evidence for faeces input of ruminants. The steroid patterns and ratios indicate a negligible input of human faeces on the Ullafelsen. In conclusion, our results reflect a strong faeces input by livestock, rather than by humans as found for other Anthrosols such as Amazonian dark earths. Further studies need to focus on the question of the exact timing of faeces deposition.