Urinary Steroid Research Articles

Adrenocortical Tumors and Pheochromocytoma/Paraganglioma Initially Mistaken as Neuroblastoma-Experiences From the GPOH-MET Registry.

In children and adolescents, neuroblastoma (NBL), pheochromocytoma (PCC), and adrenocortical tumors (ACT) can arise from the adrenal gland. It may be difficult to distinguish between these three entities including associated extra-adrenal tumors (paraganglioma, PGL). Precise discrimination, however, is of crucial importance for management. Biopsy in ACT or PCC is potentially harmful and should be avoided whenever possible. We herein report data on 10 children and adolescents with ACT and five with PCC/PGL, previously mistaken as NBL. Two patients with adrenocortical carcinoma died due to disease progression. Two (2/9, missing data in one patient) patients with a final diagnosis of ACT clearly presented with obvious clinical signs and symptoms of steroid hormone excess, while seven patients did not. Blood analyses indicated increased levels of steroid hormones in one additional patient; however, urinary steroid metabolome analysis was not performed in any patient. Two (2/10) patients underwent tumor biopsy, and in two others tumor rupture occurred intraoperatively. In 6/10 patients, ACT diagnosis was only established by a reference pediatric pathology laboratory. Four (4/5) patients with a final diagnosis of PCC/PGL presented with clinical signs and symptoms of catecholamine excess. Urine tests indicated possible catecholamine excess in two patients, while no testing was carried out in three patients. Measurements of plasma metanephrines were not performed in any patient. None of the five patients with PCC/PGL received adrenergic blockers before surgery. In four patients, PCC/PGL diagnosis was established by a local pathologist, and in one patient diagnosis was revised to PGL by a pediatric reference pathologist. Genetic testing, performed in three out of five patients with PCC/PGL, indicated pathogenic variants of PCC/PGL susceptibility genes. The differential diagnosis of adrenal neoplasias and associated extra-adrenal tumors in children and adolescents may be challenging, necessitating interdisciplinary and multidisciplinary efforts. In ambiguous and/or hormonally inactive cases through comprehensive biochemical testing, microscopical complete tumor resection by an experienced surgeon is vital to preventing poor outcome in children and adolescents with ACT and/or PCC/PGL. Finally, specimens need to be assessed by an experienced pediatric pathologist to establish diagnosis.

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.

Heritability of Urinary Amines, Organic Acids, and Steroid Hormones in Children.

Variation in metabolite levels reflects individual differences in genetic and environmental factors. Here, we investigated the role of these factors in urinary metabolomics data in children. We examined the effects of sex and age on 86 metabolites, as measured on three metabolomics platforms that target amines, organic acids, and steroid hormones. Next, we estimated their heritability in a twin cohort of 1300 twins (age range: 5.7–12.9 years). We observed associations between age and 50 metabolites and between sex and 21 metabolites. The monozygotic (MZ) and dizygotic (DZ) correlations for the urinary metabolites indicated a role for non-additive genetic factors for 50 amines, 13 organic acids, and 6 steroids. The average broad-sense heritability for these amines, organic acids, and steroids was 0.49 (range: 0.25–0.64), 0.50 (range: 0.33–0.62), and 0.64 (range: 0.43–0.81), respectively. For 6 amines, 7 organic acids, and 4 steroids the twin correlations indicated a role for shared environmental factors and the average narrow-sense heritability was 0.50 (range: 0.37–0.68), 0.50 (range; 0.23–0.61), and 0.47 (range: 0.32–0.70) for these amines, organic acids, and steroids. We conclude that urinary metabolites in children have substantial heritability, with similar estimates for amines and organic acids, and higher estimates for steroid hormones.

Urinary steroid metabolomics for adrenocortical cancer diagnosis. Comparison of gas chromatography mass spectrometry to liquid chromatography mass spectrometry

Urinary steroid metabolomics for adrenocortical cancer diagnosis. Comparison of gas chromatography mass spectrometry to liquid chromatography mass spectrometry

Urine steroid profiling as a tool for the treatment evaluation assessment in patients with adrenocortical carcinoma

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Specific characteristics of metabolomics as assessed by gas chromatography-mass spectrometry in patients with adrenocortical cancer and with adrenal incidentalomas in congenital adrenal hyperplasia

Background: Prolonged episodes of uncontrolled congenital adrenal hyperplasia (CAH) have been shown to result in the occurrence of secondary adrenal neoplasms. Prevalence of adrenal incidentalomas in the patients with 21-hydroxylase deficiency ranges from 11% to 82%. As assessed by gas chromatography-mass spectrometry (GC-MS), patients with adrenocortical cancer (ACC) have increased level of steroid hormone precursors due to decreased activity of adrenal steroidogenesis enzymes, mainly that of 21-hydroxylase and 11-hydroxylase. It seems relevant to compare the specific characteristics of steroid metabolism by GC-MS in ACC patients and in patients with adrenal incidentalomas and CAH associated with 21-hydroxylase deficiency (21-OHD).
 Aim: To identify (by GC-MS) common abnormalities in steroid metabolism and differential diagnostic biomarkers in ACC patients and CAH patients with 21-OHD and adrenal masses.
 Materials and methods: The study included 41 patients with adrenal cortex neoplasms aged 18 to 65 years without clinical and laboratory signs of endogenous hypercortisolism. Twenty three (23) patients had non-metastatic ACC and 18 patients had CAH due to 21-OHD. The control group included 26 healthy blood donors aged 20 to 59 years. Urine steroid profiles were measured by GC-MS with a gas chromatograph-mass spectrometer (Shimadzu GCMS-QP2020).
 Results: In the ACC patients, there was an increase in urinary excretion of tetrahydro-11-deoxycortisol, dehydroepiandrosterone, androstenediol-17, etiocholanolone, pregnenediol, and 3,16,20-pregnenetriol (3,16,20-dP3), as well as a decrease in the 3,16,20-dP3/3,16,20-dP3 ratio, compared to the values in the patients with CAH due to 21-OHD. Compared to the healthy control, 21-hydroxylase, 11-hydroxylase, 5-reductase and 11-hydroxysteroid-dehydrogenase (11-HSDH) type 2 activities were lower. Compared to the ACC patients, those with CAH due to 21-OHD had higher urinary excretion of 11-oxo-pregnanetriol (11-oxo-P3) and 21-deoxy-tetrahydrocortisol and lower 5-THF+5-THF+THE)/11-oxo-P3 ratio of 9.0, determination of 11-oxo-dP3, signs higher 5-reductase activity and lower 11-HSDH type 1 activity. The ACC patients and the patients with CAH due to 21-OHD had common abnormalities of steroid metabolism, such as lower activities of 21-hydroxylase, 3-hydroxysteroid-dehydrogenase and 11-hydroxylase, and no differences in urinary excretion of a number of ACC biomarkers (androgens, pregnanediol, and 5-ene-pregnenes).
 Conclusion: The assessment of urinary excretion of androgens, progestagens, and glucocorticoids by GC-MS made it possible to identify common abnormalities in steroid metabolism in the patients with ACC and CAH due to 21-OHD, which confirms the role of disordered steroidogenesis in the formation of adrenocortical tumors.

Sex steroid hormones in urinary exosomes as biomarkers for the prediction of prostate cancer

BackgroundAlthough they are involved in the progression of PCa, the use of sex steroid hormones in urinary exosomes as biomarkers for PCa remains obscure. Here, the potential use of sex steroid hormones in urinary exosomes as biomarkers was investigated for the prediction of early-stage PCa to assist in clinical diagnosis. MethodsTwo hundred and eighty-six participants were randomly recruited, 231 patients with PCa and 55 healthy controls. According to their Gleason scores (GSs), the patients with PCa were divided into two groups, mild PCa (GS6) (n = 116) and severe (≥GS7) group (n = 115). The concentrations of 8 sex steroid hormones in urinary exosomes were quantitated using liquid chromatography tandem mass spectrometry with atmospheric pressure chemical ionization (LC-APCI–MS/MS). ResultsThe results showed that the levels of 7 out of 8 sex steroids including dehydroepiandrosterone (DHEA), dehydroepiandrosteronesulfate (DHEAS), androstenedione (A4), testosterone (T), progesterone (P), dihydrotestosterone (DHT), and estrone (E1), but not estradiol (E2) in urinary exosomes, were not only distinguished the PCa patients from healthy controls, can also differentiate between patients with mild and severe PCa. Of the 8 selected urinary exosomal biomarkers, DHEA, DHEAS, T, and DHT were finally screened further to build the regression model, and the detection method of the 4 biomarkers-combined achieved an area under the ROC curve (AUC) of 0.854 and predictive accuracy of 78.2%. ConclusionOur data showed the use of exosomal sex steroids in urine could be as biomarkers for predicting PCa for the first time. This finding would supply a novel insight for PCa diagnosis.

Quantification of urinary steroids by supported liquid extraction with GC-MS/MS: Unravelling cyclic fluctuations of steroid profiling in regular menstrual cycle

A method for simultaneous quantification of urinary steroids has been developed using supported liquid extraction technique in combination with gas chromatography-triple quadruple tandem mass spectrometry. The supported liquid extraction technique could enrich low-concentration analytes by reducing matrix interference, hence the technique offered better extraction efficiency compared to conventional preparation methods. The method was shown to be linear (r2 > 0.99) in the range of concentrations for all steroids with accuracy ranging from 88.0% to 115.6%. The intra- and inter-day precision, expressed as relative standard deviation, were both below 11.9%. The lower limits of quantification were between 1 and 50 ng·mL−1, and the limits of detection were between 0.5 and 2.5 ng·mL−1, which were suitable for the detection of steroids. The validated method was subsequently applied for analyzing urinary steroid profiling in regular menstrual cycle. The cyclic fluctuations and positive correlations between the steroids in different phases were observed. The results indicated that the proposed method was an effective tool for revealing hormonal fluctuations during menstrual cycle in complex biological samples.

Long-Term Follow-Up of Three Family Members with a Novel NNT Pathogenic Variant Causing Primary Adrenal Insufficiency.

Nicotinamide nucleotide transhydrogenase (NNT) deficiency causes primary adrenal insufficiency (PAI) and possibly some extra-adrenal manifestations. A limited number of these patients were previously described. We present the clinical and genetic characteristics of three family members with a biallelic novel pathogenic variant in the NNT gene. The patients were followed until the ages of 21.6, 20.2, and 4.2 years. PAI was diagnosed in the eldest two brothers after an Addisonian crisis and the third was diagnosed at the age of 4.5 months in the asymptomatic stage due to the genetic screening of family members. Whole exome sequencing with a targeted interpretation of variants in genes related to PAI was performed in all the patients. The urinary steroid metabolome was determined by gas chromatography–mass spectrometry in the asymptomatic patient. The three patients, who were homozygous for c.1575dup in the NNT gene, developed isolated glucocorticoid deficiency. The urinary steroid metabolome showed normal excretion of cortisol metabolites. The adolescent patients had slow pubertal progression with low–normal testicular volume, while testicular endocrine function was normal. Bone mineral density was in the range for osteopenia in both grown-up siblings. Echocardiography revealed no structural or functional heart abnormalities. This article is among the first with a comprehensive and chronologically-detailed description of patients with NNT deficiency.

Susceptibility to Adrenal Crisis Is Associated With Differences in Cortisol Excretion in Patients With Secondary Adrenal Insufficiency.

ObjectiveTo compare cortisol pharmacokinetics and pharmacodynamics mapped through several glucocorticoid sensitive pathways in patients on hydrocortisone substitution with or without an adrenal crisis.DesignA post-hoc analysis of a previously conducted randomized controlled trial in patients with secondary adrenal insufficiency examining the effects of 2 weight-adjusted hydrocortisone doses.MethodsComparisons were primarily made on a hydrocortisone dose of 0.2-0.3 mg/kg/day for plasma cortisol and cortisone, 24-hour urinary steroid profile, the glucocorticoid sensitive tryptophan-kynurenine pathway, the renin-angiotensin-aldosterone system and aspects of quality of life. Variables of interest were also analyzed on the hydrocortisone dose of 0.4-0.6 mg/kg/day.ResultsOut of 52 patients, 9 (17%) experienced at least one adrenal crisis (AC+ group) and 43 did not develop an adrenal crisis (AC- group) during an observation period of 10 years. 24-hour urinary excretion of cortisol and cortisone were lower in the AC+ group (0.05 [IQR 0.03; 0.05] vs. 0.09 [0.05; 0.12] µmol/24h, P=0.01and 0.13 [0.10; 0.23] vs. 0.24 [0.19; 0.38] µmol/24h, P=0.04, respectively). No differences in pharmacokinetics of cortisol were observed. Kynurenine concentrations were higher in the AC+ group (2.64 [2.43; 3.28] vs. 2.23 [1.82; 2.38] µmol/L, P=0.03) as was general fatigue (Z-scores 1.02 [-0.11; 1.42] vs. -0.16 [- 0.80; 0.28], P=0.04). On the higher hydrocortisone dose urinary excretion of cortisol and cortisone was still significantly lower between the AC- and AC + group. The differences in glucocorticoid sensitive variables disappeared.ConclusionPatients susceptible to an adrenal crisis demonstrated differences in cortisol and cortisone excretion as well as in pharmacodynamics when compared to patients who did not experience an adrenal crisis, suggesting a biological predisposition in certain patients for the development of an adrenal crisis.

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.

Urinary cortisol metabolites are reduced in MDR1 mutant dogs in a pilot targeted GC-MS urinary steroid hormone metabolome analysis.

P-glycoprotein (P-gp) is the gene product of the multidrug resistance gene (MDR1, syn. ABCB1) that normally restricts the transfer of cortisol across the blood-brain barrier. In the absence of P-gp, cortisol access to the hypothalamus is increased and, by feedback inhibition, this finally leads to lower endogenous plasma cortisol levels in dogs with homozygous nt230(del4) MDR1mutation (MDR1-/- mutant dogs). While a previous study only focused on plasma cortisol levels, the present study used urinary steroid hormone metabolites to analyze cortisol metabolism in MDR1-/- mutant dogs. Morning void urine was collected from 23MDR1-/- mutant and 16MDR1+/+ normal dogs and was subjected to targeted GC-MS steroid hormone metabolome analysis. Seven cortisol metabolites, cortisol itself, and 13 other steroid metabolites were detected. In general, all cortisol metabolites were lower in the urine of the MDR1-/- mutant dogs, with allo-tetrahydro-cortisol and β-cortol reaching the level of significance. In addition, 11-keto-pregnanetriol levels were significantly lower in the urine of the MDR1-/- mutant dogs, indicating that also the 17alpha-OH-progesterone-derived metabolism was altered. In conclusion, the present study provides the first steroid hormone metabolome analysis in the urine of MDR1-/- mutant dogs. Significant differences in the steroid metabolome of MDR1-/- mutant dogs point to a significant role of P-gp for cortisol metabolism and excretion and so indirectly also for hypothalamic-pituitary-adrenal axis regulation in dogs.

Ketoconazole- and Metyrapone-Induced Reductions on Urinary Steroid Metabolites Alter the Urinary Free Cortisol Immunoassay Reliability in Cushing Syndrome.

IntroductionTwenty-four-hour urinary free cortisol (24h-UFC) is the most used test for follow-up decision-making in patients with Cushing syndrome (CS) under medical treatment. However, 24h-UFC determinations by immunoassays (IA) are commonly overestimated because of steroid metabolites’ cross-reaction. It is still uncertain how ketoconazole (KTZ)- and metyrapone (MTP)-induced changes on the urinary steroid metabolites can alter the 24h-UFC*IA determinations’ reliability.Methods24h-UFC was analyzed by IA and gas chromatography-mass spectrometry (GC-MS) in 193 samples (81 before treatment, 73 during KTZ, and 39 during MTP) from 34 CS patients. In addition, urinary steroidome was analyzed by GC-MS on each patient before and during treatment.ResultsBefore treatment, 24h-UFC*IA determinations were overestimated by a factor of 1.75 (95% CI 1.60–1.94) compared to those by GC-MS. However, during KTZ treatment, 24h-UFC*IA results were similar (0.98:1) to those by GC-MS (95% CI, 0.83–1.20). In patients taking MTP, IA bias only decreased 0.55, resulting in persistence of an overestimation factor of 1.33:1 (95% CI, 1.09–1.76). High method agreement between GC-MS and IA before treatment (R2 = 0.954) declined in patients under KTZ (R2 = 0.632) but not in MTP (R2 = 0.917). Upper limit normal (ULN) reductions in patients taking KTZ were 27% larger when using 24h-UFC*IA compared to 24h-UFC*GC-MS, which resulted in higher false efficacy and misleading biochemical classification of 15% of patients. Urinary excretion changes of 22 urinary steroid metabolites explained 86% of the 24h-UFC*IA interference. Larger urinary excretion reductions of 6β-hydroxy-cortisol, 20α-dihydrocortisol, and 18-hydroxy-cortisol in patients with KTZ elucidated the higher 24h-UFC*IA bias decrement compared to MTP-treated patients.ConclusionKTZ and MTP alter the urinary excretion of IA cross-reactive steroid metabolites, thus decreasing the cross-reactive interference of 24h-UFC*IA determinations present before treatment. Consequently, this interference reduction in 24h-UFC*IA leads to loss of method agreement with GC-MS and high risk of overestimating the biochemical impact of KTZ and MTP in controlling CS because of poor reliability of reference ranges and ULN.

Implementation of a marker substance for monitoring in situ 17-keto modifications in endogenous steroids caused by microbiological contamination.

The urinary steroid profile established for the monitoring of eventual testosterone or testosterone precursor application by athletes includes concentrations and ratios of various endogenously produced steroidal hormones and metabolites. Due to enzymatic activities in urine specimens, the concentrations of these endogenous steroids and consequently their ratios may alter, leading to potential misinterpretation of analytical results. Microbiological contamination in athletes' urine samples can occur due to urinary tract infections or due to contamination by the non-sterile sample collection conditions. Depending on the duration of transportation of urine samples, the transport and storage conditions may favour microorganisms' growth, and therefore, the enzymatic activity can be accelerated. Degradation effects on endogenous steroids caused by microorganisms have been observed, such as hydrolysis of steroid conjugates, increase of testosterone in the free fraction or modification of the steroid structure by oxidoreductive reactions. The World Anti-Doping Agency (WADA) implemented criteria to check for signs of microbial degradation in a technical document dealing with the detection, analysis and reporting of endogenous androgenic anabolic steroids (TD EAAS) in urine samples. During the endogenous steroid profile confirmation procedures (CPs) of the WADA accredited Seibersdorf Laboratory, significant differences in the concentrations of markers of the steroid profile were observed compared to the initial testing procedures (ITPs). The changes in concentrations of the urinary steroid profile were attributed to the reduction of the 17-keto group to a 17β-hydroxy group caused by increased enzymatic activity during the hydrolysis step. In order to monitor the 17-keto reduction activity in athletes' urine specimens, possible marker substances containing a 17-keto group were synthesised and added in the internal standards mixture (ISTD) of the ITP. The presence of the reduced 17β-hydroxy form of the marker substance indicated enzymatic activity leading to 17-keto reduction reactions. The substance 3β-ethoxy-5α-androstane-17-one was defined to be suitable to indicate 17-keto reduction reactions occurring during hydrolysis carried out at moderate temperatures.

Non-invasive metabolomics biomarkers of production efficiency and beef carcass quality traits

The inter-cattle growth variations stem from the interaction of many metabolic processes making animal selection difficult. We hypothesized that growth could be predicted using metabolomics. Urinary biomarkers of cattle feed efficiency were explored using mass spectrometry-based untargeted and targeted metabolomics. Feed intake and weight-gain was measured in steers (n = 75) on forage-based growing rations (stage-1, 84 days) followed by high-concentrate finishing rations (stage-2, 84 days). Urine from days 0, 21, 42, 63, and 83 in each stage were analyzed from steers with the greater (n = 14) and least (n = 14) average-daily-gain (ADG) and comparable dry-matter-intake (DMI; within 0.32 SD of the mean). Steers were slaughtered after stage-2. Adjusted fat-thickness and carcass-yield-grade increased in greater-ADG-cattle selected in stage-1, but carcass traits did not differ between ADG-selected in stage-2. Overall 85 untargeted metabolites segregated greater- and least-ADG animals, with overlap across diets (both stages) and breed type, despite sampling time effects. Total 18-bile acids (BAs) and 5-steroids were quantified and associated with performance and carcass quality across ADG-classification depending on the stage. Stepwise logistic regression of urinary BA and steroids had > 90% accuracy identifying efficient-ADG-steers. Urine metabolomics provides new insight into the physiological mechanisms and potential biomarkers for feed efficiency.

Detection of testosterone microdosing in healthy females.

The ready detectability of synthetic androgens by mass spectrometry (MS)-based antidoping tests has reoriented androgen doping to using testosterone (T), which must be distinguished from its endogenous counterpart making detection of exogenous T harder. We investigated urine and serum steroid and hematological profiling individually and combined to determine the optimal detection model for T administration in women. Twelve healthy females provided six paired blood and urine samples over 2 weeks prior to treatment consisting of 12.5-mg T in a topical transdermal gel applied daily for 7 days. Paired blood and urine samples were then obtained at the end of treatment and Days 1, 2, 4, 7, and 14 days later. Compliance with treatment and sampling was high, and no adverse effects were reported. T treatment significantly increased serum and urine T, serum dihydrotestosterone (DHT), urine 5α-androstane-3α,17β-diol (5α-diol) epitestosterone (E), and urine T/E ratio with a brief window of detection (2-4days) as well as total and immature (medium and high fluorescence) reticulocytes that remained elevated over the full 14 posttreatment days. Carbon isotope ratio MS and the OFF score and Abnormal Blood Profile score (ABPS) were not discriminatory. The optimal multivariate model to identify T exposure combined serum T, urine T/E ratio with three hematological variables (% high fluorescence reticulocytes, mean corpuscular hemoglobin, and volume) with the five variables providing 93% correct classification (4% false positive, 10% false negatives). Hence, combining select serum and urine steroid MS variables with reticulocyte measures can achieve a high but imperfect detection of T administration to healthy females.

PHARMACEUTICAL MARKET ASSORTMENT RESEARCH OF DRUGS FOR VAGINAL USE WITH PROBIOTIC MEDICINES DETAILING

vaginal route delivery of drugs is an integral part of gynaecological diseases treatment including different genesis vulvovaginitis. The aim of this study it to analyze Ukrainian pharmaceutical market assortment of drugs for vaginal use with probiotic medicines determination. The marketing research was conducted with using of analysis, synthesis, comparison, generalization, content-analysis, statistical, graphical methods. The data of State register of medicines (as of June, 30, 2021), register of wholesale dispensing prices for medicines (as of June, 30, 2021), official web-sites LLC «Morion», «Pharmacy.online», «Compendium.online» were used as objects of the study. As a result, it is determined that as of June 30, 2021 142 medicines for vaginal use are registered on Ukrainian pharmaceutical market with dosage forms, active substances doses, condition for dispensing consideration, 100 brand names, 26 international nonproprietary names, 7 synonymous names. Structural ATC-classification analysis has shown that medicines for vaginal use are included in ATC-groups G «Genito urinary system and sex hormones» and D «Dermatologicals» The assortment macrocontour is formed, it demonstrates that 67,2% of drugs for vaginal use are involved in group G01A «Аntiinfectives and antiseptics, excluding combinations with corticosteroids». More than 80% of drugs for vaginal use are registered as solid dosage forms, and almost half of them as suppositories or pessaries. Almost 65% of medicines are foreign, and 3/4 of them contain only active substance, more than 50% are prescription only medicines. For 44,4% drugs for vaginal use the storage period of 3 years (36 months) is determined. Detailing analysis of medicines with probiotic activity has shown assortment deficiency (only 3 brand names are registered). The liquidity coefficients calculations for this brand manes don’t allow to characterize these drugs as economically affordable. The obtained results of this study inform about the domestic drugs for vaginal use with probiotic activity development relevance, especially in pessaries as dominant dosage form on the pharmaceutical market.

Toward Routine Analysis of Anabolic Androgenic Steroids in Urine Using Ion Mobility-Mass Spectrometry.

Anabolic androgenic steroids (AAS) make up one of the most prevalent classes of performance-enhancing drugs banned by the World Anti-Doping Agency (WADA) due to the competitive advantage they can afford athletes. Mass spectrometry-based methods coupled with chromatographic separations have become the gold standard for AAS analysis because of the superior sensitivity and selectivity provided. However, emerging analytical techniques including ion mobility spectrometry (IMS) have been demonstrated in recent applications as a means to further characterize and identify potential unknowns while simultaneously delivering improved sensitivity by filtering noise. Herein we outline the next crucial steps in bringing IMS to the routine drug testing workflow by combining it with established chromatographic and mass spectrometry methods (i.e., LC-IM-MS) for the detection of AAS in human urine. In addition to robust measurement of collision cross sections which can be used for identification purposes, functional group microtrends provide a structural basis on which to elucidate the structure of future novel anabolic agents. Lastly, the developed workflow is tested by analysis of testosterone in a realistic matrix (human urine) and demonstrates a limit of detection of 524 pg/mL, which surpasses the WADA Minimum Required Performance Levels for anabolic steroids. This work is expected to pave the way toward routine incorporation of IMS into analytical drug testing workflows to augment both qualitative and quantitative measure of performance enhancing drugs in the future.

Adrenal Steroid Metabolism and Blood Pressure in 5- to 7-Year-Old Children Born Preterm as Compared to Peers Born at Term.

Background: Previous studies indicated preterm birth to be a risk factor for hypertension in adolescence and adulthood. However, studies in children investigating the underlying mechanisms are scarce.Objective: We hypothesized children born preterm to have higher excretion of cortisol and/or androgen metabolites per day concomitantly with higher blood pressure as compared to peers born at term. We thus aimed to compare urinary steroid profiles and blood pressure between 5- to 7-year-old children born preterm and peers born at term. Furthermore, aldosterone precursor excretion per day was compared between both groups.Methods: Blood pressure was measured in 236 children (preterms n = 116; gestational age 29.8 ± 2.6 (30; 24–33) weeks [mean ± standard deviation (median; range)]) using an automatic oscillometric device. Urinary steroid profiles were determined in 24-h urine samples (preterms n = 109; terms n = 113) using gas chromatographic-mass spectrometric analysis. To assess excretion of cortisol and androgen metabolites per day, major cortisol and androgen metabolites were summed, respectively. To assess aldosterone excretion per day tetrahydrocorticosterone, 5α-tetrahydrocorticosterone, and tetrahydro-11-deydrocorticosterone were summed.Results: Multiple regression analyses showed prematurity to be associated with systolic but not with diastolic blood pressure. When adjusted for potential confounders (prematurity, gender, age at day of examination, being born small for gestational age, breastfeeding, accelerated weight gain during infancy, family history of cardiovascular disease, parental hypertension, and body mass index) prematurity was shown to be associated with an increase in systolic blood pressure by 2.87 mmHg (95% confidence interval 0.48–5.27; p = 0.02). Cortisol, androgen metabolite, and aldosterone precursor excretion per day were not higher in individuals born preterm. In contrast to our hypothesis, multiple regression analysis showed prematurity to independently decrease cortisol and aldosterone precursor excretion per day (p < 0.001 and 0.04, respectively).Conclusion: This study provides further evidence for systolic blood pressure to be higher after preterm birth as early as at the age of 5 to 7 years. However, this seems not to be explained by elevated excretion of cortisol and/or androgen metabolites.

Study of urine steroid profiles by gas chromatography-mass spectrometry in patients with adrenocortical cancer in the course of treatment

Background: Adrenocortical carcinoma (ACC) is a rare and aggressive disease. There are only few studies evaluating the diagnostic value of gas chromatography-mass spectrometry (GC-MS) for detection of ACC recurrence after surgery. It is necessary to conduct an in-depth study to search for the most informative markers of the disease relapse.Aim: To study urine steroid metabolism by GC-MS during treatment to identify early signs of metastatic disease and relapse.Materials and methods: Thirty nine (39) ACC patients were examined before and after surgery, in the early postoperative period (&lt; 1 year) and late postoperative period (at 2 to 5 years). Ten (10) patients were disease-free at less than 1 year after surgery. Twenty nine (29) patients had metastases in lungs and other organs: 14, within 1 year after surgery, and 15, at 2 to 5 years. The control group included 25 patients with nonfunctioning adrenocortical adenomas (NAA) without malignant characteristics at histological examination. Urine steroid profiles were assessed with a gas chromatograph-mass spectrometer Shimadzu GCMS-QP2020.Results: As assessed by GC-MS, 16 major ACC biomarkers were found before surgery, including etiocholanolone, dehydroepiandrosterone (DHEA) and its metabolites, pregnanediol, pregnanetriol, 5-ene-pregnenes, and tetrahydro-11-deoxycortisol (THS). Their urine excretion was increased compared to that in the patients with NAA (р &lt; 0.002). A non-classic 5-ene-pregnene, 3β,16,20-pregnenetriol (3β,16,20-dP3), was identified, with its urine excretion of &gt; 500 mcg/day that was typical for ACC patients. After surgery, decreased urinary excretion of THS (р &lt; 0.0001) and 3β,16,20-dP3 (р &lt; 0.0001), increased 3α,16,20-dP3/3β,16,20-dP3 ratio (р = 0.003), compared to those before surgery, were indicative of the absence of any metastases. No difference of urine THS excretion and 3α,16,20-dP3/3e,16,20-dP3 ratio from the corresponding values before surgery (p &gt; 0.05) is a sign of metastatic diseases in the ACC patients at less than 1 year after the surgery, of the disease relapse at 2 to 5 years, and of the disease relapse after chemotherapy. In addition, in the ACC patients with metastatic disease within 1 year after surgery, increased progestogen urine excretion was found. Urine excretion of DHEA and its metabolites in the patients with the disease relapse after chemotherapy was not different from those in the ACC patients before surgery (p &gt; 0.05).Conclusion: Determination of urine excretion of THS, DHEA and its metabolites, etiocholanolone, 5-ene-pregnenes, 3β,16,20-dP3, and 3α,16,20-dP3/3β,16,20-dP3 ratio by GC-MS is of utmost importance in the monitoring of treatment for ACC and early diagnosis of the disease progression.