Effect of oxidizing adulterants on human urinary steroid profiles

Steroid profiling is the most versatile and informative technique adapted by doping control laboratories for detection of steroid abuse. The absolute concentrations and ratios of endogenous steroids including testosterone, epitestosterone, androsterone, etiocholanolone, 5α-androstane-3α,17β-diol and 5β-androstane-3α,17β-diol constitute the significant characteristics of a steroid profile. In the present study we report the influence of various oxidizing adulterants on the steroid profile of human urine. Gas chromatography–mass spectrometry analysis was carried out to develop the steroid profile of human male and female urine. Oxidants potassium nitrite, sodium hypochlorite, potassium permanganate, cerium ammonium nitrate, sodium metaperiodate, pyridinium chlorochromate, potassium dichromate and potassium perchlorate were reacted with urine at various concentrations and conditions and the effect of these oxidants on the steroid profile were analyzed. Most of the oxidizing chemicals led to significant changes in endogenous steroid profile parameters which were considered stable under normal conditions. These oxidizing chemicals can cause serious problems regarding the interpretation of steroid profiles and have the potential to act as masking agents that can complicate or prevent the detection of the steroid abuse.     

Expression of hepatic 3β-hydroxysteroid dehydrogenase and sulfotransferase 2A1 in entire and castrated male pigs

The present study investigated the effect of surgical (SC) and immunological castration on the steroid metabolizing enzymes 3β-hydroxysteroid dehydrogenase (3β-HSD) and sulfotransferase 2A1 (SULT2A1) in male pigs. Thirty-two male pigs were divided in four groups; in one group the pigs were SC before the age of 7 days, two groups were injected with Improvac(?) a vaccine against gonadotropin releasing hormone (immunological castration), while the pigs in the last group remained entire males (EMs). Immunological castration was in one group performed by vaccine injection at ages 11 and 14 weeks, while the other group received injections at ages 17 and 21 weeks. Plasma, adipose and liver tissue were collected at the time of slaughter. Plasma was analyzed for concentrations of testosterone and oestradiol. The adipose tissue was analyzed for the concentration of androstenone, while the liver tissue was analyzed for mRNA and protein expression of 3β-HSD and SULT2A1. Independent of method, all castrated pigs showed greater mRNA and protein expression of 3β-HSD and lower levels of all steroids in plasma compared with EMs. Moreover, there was a strong correlation between mRNA and protein expression of 3β-HSD and steroid levels. The same was not valid for expression of SULT2A1. It is concluded that steroid levels can increase expression of the steroid metabolizing enzyme 3β-HSD and thereby influence steroid metabolism, e.g. of androstenone.     

Activated p38 MAPK in Peripheral Blood Monocytes of Steroid Resistant Asthmatics

Steroid resistance is a significant problem in management of chronic inflammatory diseases, including asthma. Accessible biomarkers are needed to identify steroid resistant patients to optimize their treatment. This study examined corticosteroid resistance in severe asthma. 24 asthmatics with forced expiratory volume in one second of less then 80% predicted were classified as steroid resistant or steroid sensitive based on changes in their lung function following a week of treatment with oral prednisone. Heparinised blood was collected from patients prior to oral prednisone administration. Phosphorylated mitogen activated kinases (MAPK) (extracellular regulated kinase (ERK), p38 and jun kinase (JNK)) were analyzed in whole blood samples using flow cytometry. Activation of phospho-p38 MAPK and phospho-mitogen- and stress-activated protein kinase 1 (MSK1) in asthmatics’ peripheral blood mononuclear cells (PBMC) were confirmed by Western blot. Dexamethasone suppression of the LPS-induced IL-8 mRNA production by steroid resistant asthmatics PBMC in the presence of p38 and ERK inhibitors was evaluated by real time PCR. Flow cytometry analysis identified significantly stronger p38 phosphorylation in CD14⁺ monocytes from steroid resistant than steroid sensitive asthmatics (p = 0.014), whereas no difference was found in phosphorylation of ERK or JNK in CD14⁺ cells from these two groups of asthmatics. No difference in phosphorylated p38, ERK, JNK was detected in CD4⁺, CD8⁺ T cells, B cells and NK cells from steroid resistant vs. steroid sensitive asthmatics. P38 MAPK pathway activation was confirmed by Western blot, as significantly higher phospho-p38 and phospho-MSK1 levels were detected in the PBMC lysates from steroid resistant asthmatics. P38 inhibitor significantly enhanced DEX suppression of LPS-induced IL-8 mRNA by PBMC of steroid resistant asthmatics. This is the first report demonstrating selective p38 MAPK pathway activation in blood monocytes of steroid resistant asthmatics, suggesting that p38 and MSK1 phosphorylation can serve as blood biomarkers of steroid resistance.     

Estrogen, progesterone receptors and proliferating activity evaluated by immunocytochemistry in breast cancer.

The correlation of the most important prognostic indicators was evaluated in 75 breast cancer cases. Estrogen-progesterone receptors and proliferating activity were analyzed by immunocytochemical methods (ER-ICA, PR-ICA, Ki-67). Both steroid receptors were inversely correlated with the proliferating activity (ER-ICA vs Ki-67, p less than 0.003; PR-ICA vs. Ki-67, p less than 0.0001). No correlation was found between steroid receptors or cell kinetics and tumor size or lymph node status. These findings confirm the relevance of biochemical and kinetic parameters as independent markers in breast cancer and suggest a routine use of the simple immunocytochemical methods in assessing the biological behavior of tumors.     

Expression of sex steroid receptors and IGF-1 mRNA in breast tissue--effects of hormonal treatment

The mechanisms behind increased breast tissue proliferation and a possibly increased breast cancer risk in women using hormonal contraception (HC) and hormonal replacement therapy (HRT) are incompletely understood. We analyzed breast tissue from 20 premenopausal and seven postmenopausal women undergoing reduction mammoplasties for estrogen receptor (ER) and progesterone receptor (PR) content as well as mRNA levels for ER, PR and insulin-like growth factor-1 (IGF-1). The receptor values were correlated to IGF-1 mRNA concentrations and levels of steroid and peptide hormones and SHBG. In women using HC, we found significantly lower ER values (p=0.02) but non-significantly lower ER mRNA levels compared to those in naturally cycling women. PR and PR mRNA were no different. Women on HC displayed a higher breast tissue proliferation (p=0.05) expressed as Ki-67, MIB-1 positivity, which was correlated with IGF-1 mRNA (r_s=0.82, p=0.04). Since the concentration of sex steroid receptors in breast tissue is comparatively low and steroid receptors are down-regulated during hormonal treatment, mechanisms other than direct sex steroid receptor action are likely to be present. Our results suggest a role for IGF-1 in the proliferative response of breast tissue during exogenous hormonal treatment.     

Effect of anabolic steroids on overloaded and overloaded suspended skeletal muscle

The purpose of this study was to ascertain whether anabolic steroids act synergistically with functional overload in terms of increasing muscle weight and subcellular protein content of normal overloaded and suspended overloaded rodent plantaris muscle. Female rats were randomly assigned to six groups (7 rats/group) for 6 wk: 1) normal control (NC), 2) overload (OV), 3) overload steroid (OV-S), 4) normal suspended (N-SUS), 5) overload suspended (OV-SUS), and 6) overload suspended steroid (OV-SUS-S). Rats receiving anabolic steroid were administered 0.3 mg nandrolone decanoate (Deca-Durabolin) per day. Relative to control values, overload induced 1) sparing of muscle weight of the OV-SUS group as well as larger absolute and normalized (mg muscle/g body wt) muscle weight of the OV group (P less than 0.05), 2) greater protein content (mg/muscle, P less than 0.05), and 3) an increased relative expression of slow myosin in both the OV and OV-SUS groups (P less than 0.05). Although anabolic steroid treatment of overload animals (OV-S) did not alter further the pattern of response of any parameter analyzed for the OV group, it did induce larger absolute and normalized muscle weight (P less than 0.05) as well as a greater protein content (mg/muscle, P less than 0.05) of the OV-SUS-S group compared with control values. However, anabolic steroid treatment did not alter the pattern of isomyosin expression observed in the overload (OV-S vs. OV) or overload suspended (OV-SUS-S vs. OV-SUS) groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Correlation in inflammatory fibroblasts between the number of glucocorticoid receptors and PGE2 release

The anti-inflammatory effects of glucocorticoids are mediated through steroid receptor occupancy and there is a significant correlation between the extent of receptor saturation and the extent of the biological effects. In a previously published study, we found that the number of these receptors was higher in inflammatory fibroblasts than in quiescent ones. PGE2 release, measured at the same time as the number of steroid receptors, was higher when the cells were from inflammatory tissue. Our aim, in the present study, was to determine whether the PGE2 released by cells during inflammatory processes could participate in increasing the number of steroid receptors. Fibroblasts obtained from rat quiescent subcutaneous connective tissue and granulomas were subcultured in monolayers. The specific binding of [3H]dexamethasone was assessed and analyzed by a method described by Kalimi et al. After a freeze-thaw cycle, we observed a decrease in the number of receptors in inflammatory fibroblasts. When the frozen and thawed fibroblasts were subcultured in the presence of PGE2 (10(-8) M), the number of receptors was enhanced in fibroblasts from inflammatory tissue. Cycloheximide (3 X 10(-7) M) prevented this increase. The release of PGE2 decreased after freezing and then increased simultaneously with the number of receptors in inflammatory cells. These findings suggest that PGE2 may play a role in regulating steroid effects on fibroblast function.     

Maternal steroids and contaminants in common tern eggs: a mechanism of endocrine disruption?

We looked for evidence for the hypothesis that exposure of female birds to polychlorinated biphenyls (PCBs) results in alteration of blood steroid hormone concentrations and alters subsequent hormone transfer of steroids to eggs. Eggs of three-egg clutches were collected from a PCB-exposed common tern (Sterna hirundo) colony (Ram Island, Buzzards Bay, MA, USA) and from a relatively clean colony (Bodkin Island, Chesapeake Bay, MD, USA), and were analyzed for concentrations of organochlorine contaminants and steroid hormones (17beta-estradiol, 5alpha-dihydrotestosterone, testosterone and androstenedione). There was no relationship between total PCBs and steroid concentrations considering all eggs together, considering eggs of different laying order or considering differences between sequentially laid eggs in a clutch. Similarly, concentrations of di- and tri-chlorinated biphenyls and steroids in eggs were not related. The concentrations of PCBs, mercury and selenium were below estimated thresholds for toxicity to embryos. Maternal steroids, except estradiol, were present in yolk of all eggs, with increasing concentrations in the second and third eggs laid. Our data provided no evidence for a maternal toxicological event that might alter the amount of maternal steroid hormone transferred to eggs.     

In situ estrogen metabolism in proliferative endometria from untreated women with polycystic ovarian syndrome with and without endometrial hyperplasia

The aim of the present investigation was to study whether the endocrinological status of women bearing polycystic ovarian syndrome (PCOS) affects the endometrial in situ steroid metabolism. For this purpose, we evaluated the mRNA levels (RT-PCR), and the activity of steroid metabolic enzymes: P450 aromatase, steroid sulfatase (STS), estrogen sulfotransferase (EST) and 17beta-hydroxysteroid dehydrogenase (17beta-HSD) in 23 samples of normal endometria (CE), 18 PCOS endometria without treatment (PCOSE), 10 specimens from PCOS women with endometrial hyperplasia (HPCOSE), and 7 endometria from patients with endometrial hyperplasia not associated to PCOS (EH). The data showed lower levels of STS mRNA for PCOSE and HPCOSE (p<0.05, p<0.01, respectively) and of EST for HPCOSE and EH compared to control (p<0.05). However, higher levels for EST mRNA were obtained in PCOSE (p<0.05) versus CE. The mRNA and protein levels for P450 aromatase were undetectable in all analyzed endometria. The relationship between the activities of STS and EST was lower in PCOSE and HPCOSE (p<0.05) versus CE. The ratio between the mRNA from 17beta-HSD type 1/type 2 was higher in PCOSE (p<0.05), whereas, a diminution in the 17beta-HSD type 2 activity was observed in PCOSE (p<0.05). These results indicate that the activity of enzymes related to the steroid metabolism in analyzed PCOSE differ from those found in the CE. Consequently, PCOSE may present an in situ deregulation of the steroid metabolism.     

Adolescent anabolic/androgenic steroids: Aggression and anxiety during exposure predict behavioral responding during withdrawal in Syrian hamsters (Mesocricetus auratus)

In the U.S. and worldwide anabolic/androgenic steroid use remains high in the adolescent population. This is concerning given that anabolic/androgenic steroid use is associated with a higher incidence of aggressive behavior during exposure and anxiety during withdrawal. This study uses pubertal Syrian hamsters (Mesocricetus auratus) to investigate the hypothesis that an inverse behavioral relationship exists between anabolic/androgenic steroid-induced aggression and anxiety across adolescent exposure and withdrawal. In the first experiment, we examined aggression and anxiety during adolescent anabolic/androgenic steroid exposure and withdrawal. Adolescent anabolic/androgenic steroid administration produced significant increases in aggression and decreases in anxiety during the exposure period followed by significant decreases in aggression and increases in anxiety during anabolic/androgenic steroid withdrawal. In a second experiment, anabolic/androgenic steroid exposed animals were separated into groups based on their aggressive response during the exposure period and then tested for anxiety during exposure and then for both aggression and anxiety during withdrawal. Data were analyzed using a within-subjects repeated measures predictive analysis. Linear regression analysis revealed that the difference in aggressive responding between the anabolic/androgenic steroid exposure and withdrawal periods was a significant predictor of differences in anxiety for both days of testing. Moreover, the combined data suggest that the decrease in aggressive behavior from exposure to withdrawal predicts an increase in anxiety-like responding within these same animals during this time span. Together these findings indicate that early anabolic/androgenic steroid exposure has potent aggression- and anxiety-eliciting effects and that these behavioral changes occur alongside a predictive relationship that exists between these two behaviors over time.     

Profile of sex steroids in healthy volunteers' urine during experiment in isolated object

The quantitative determination a number of endogenous steroids and their metabolites in urine of healthy volunteers by means of gas chromatography - mass spectrometry was performed. The dynamic of steroid profile of healthy individuals as well as possible ranges of several endogenous steroid parameters have been investigated. Samples were obtained during 105-days experiment with 6 volunteers in isolated on ground modules where were modeling the main life conditions which could influence the steroid profile: meal volume and composition, water consumption, motion activity, air composition and temperature, rate sleep - wakefulness and emotional tension. The parameters of urine steroid profile of healthy volunteer which were affected by life conditions in isolated object were revealed. The parameters of individual and group variability of steroid profile and its dependence from definite experiment conditions - change of salt consumption periods, autonomy of vital activity were detected.     

Time course of fungal removal of lipophilic extractives from Eucalyptus globulus wood

Free and esterified sitosterol, the main lipophilic constituents of eucalypt wood extractives, have been associated with the formation of pitch deposits during manufacturing of environmentally-sound paper pulp from Eucalyptus globulus wood. These, and other lipophilic compounds, were analyzed by gas chromatography-mass spectrometry in the course of wood treatments (up to 7 weeks) with four extractive-degrading fungi in order to optimize biotechnological control of pitch deposition in eucalypt pulp (with moderate loss of wood weight). In contrast to commercialized fungi used in pitch control, which are not able to degrade sitosterol, the fungi investigated in this paper produced a rapid decline of both free and esterified sterols in wood. The degradation rate of steroid hydrocarbons and squalene was moderate, and the amount of steroid ketones (probably formed during oxidative degradation of steroids) and triglycerides increased at different stages of wood treatment. Up to 95% removal of total steroids (including free and esterified sterols, steroid ketones and steroid hydrocarbons) by fungi was obtained at the end of wood treatment under the solid-state fermentation conditions used. The most promising results from the point of view of industrial applicability, however, were obtained after 1-2 weeks of treatment with either Phlebia radiata or Poria subvermispora, which enabled 70% steroid removal with a moderate wood weight loss of 1-4%.     

Purification and characterization of rat adrenal 3 beta-hydroxysteroid dehydrogenase with steroid 5-ene-4-ene-isomerase

After solubilization of rat adrenal microsomes with sodium cholate, 3 beta-hydroxysteroid dehydrogenase with steroid 5-ene-4-ene isomerase (abbreviated as steroid isomerase) activity was purified to a homogeneous state. The following characteristics of the enzyme were obtained: 3 beta-Hydroxysteroid dehydrogenase together with steroid isomerase was detected as a single protein band in SDS-polyacrylamide gel electrophoresis, where its mol. wt was estimated as 46,500. Either NAD+ or NADH was required for demonstration of steroid isomerase activity. Treatment of the enzyme with 5'-p-fluorosulfonylbenzoyladenosine, an affinity labeling reagent for NAD+-dependent enzyme, diminished both the enzyme activities.     

Neonatal urinary steroids in Smith-Lemli-Opitz syndrome associated with 7-dehydrocholesterol reductase deficiency.

The biosynthetic abnormality in Smith-Lemli-Opitz syndrome (SLOS) is a deficiency of 7-dehydrocholesterol (7DHC) reductase, the enzyme responsible for catalyzing the final step in the Kandutsch-Russell pathway for cholesterol synthesis. Because the disposition of 7DHC and 8-dehydrocholesterol [8DHC; cholesta-5,8(9)-dien-3beta-ol] produced in this syndrome is little understood, we have analyzed urine from three young infants by gas chromatography/mass spectrometry to characterize its steroid metabolites. All steroid metabolites of adrenal origin found in normal infant urine were also found in urine from the patients with SLOS but in reduced amount. Quantitatively, the major steroids in these SLOS patients were identified by mass spectrometry as homologs of normal neonatal steroids possessing an additional double bond. Generally, two forms of each steroid were present in a similar amount. Because of the markedly increased levels of 7DHC and 8DHC in SLOS, these almost certainly represented the 5,7 and 5,8(9) unsaturated forms of each metabolite. The most abundant steroids were tentatively identified as 3beta,16alpha-dihydroxy-5,7-pregnadien-20-one and 3beta,16alpha-dihydroxy-5,8(9)-pregnadien-20-one, although similar 21-hydroxylated steroids and homologs of 16alpha-hydroxy-DHEA were also found. This study shows that all enzymatic steps used by cholesterol in the DHEA synthetic pathway are also functional for 7DHC and 8DHC.     

Effect of porcine placenta steroid extract on myogenic satellite cell proliferation, transdifferentiation, and lipid accumulation

Intramuscular long-chain fatty acids (LCFAs) play an important role in energy production and initiation of mitochondrial oxidation of lipids. Herein, we report a natural porcine placenta steroid extract (PPSE) that stimulates transdifferentiation and lipid accumulation in bovine myogenic satellite cells (MSCs). The steroids hormones in PPSE were analyzed using enzyme-linked immunosorbant assay and presence of LCFA was established using gas chromatography. At 70% confluent growth, cells were treated with PPSE, LCFAs, transdifferentiation cocktail and commercially available steroid hormones. The working concentrations of all chemicals were manipulated similar to PPSE. The cells were observed for morphological changes and subjected to quantitative analysis of lipid deposition on Days 2, 4, and 6 of treatment. PPSE-treated MSCs exclusively transformed into lipid-accumulated adipose-like cells (ALCs). However, myotubes or adipocytes were formed in cells treated with other chemicals. Expression of different genes was studied to ascertain the molecular mechanism involved in ALC formation. CD36, fatty acid binding protein 4, and peroxisome proliferator-activated receptor-gamma were up-regulated. The expression of CD36 was established through immunocyto-chemical analysis. A viability assay was used to confirm the effect of PPSE on proliferation of MSCs. Hence, a natural steroid extract from porcine was found as a nontoxic mixture, which induces lipid accumulation and transdifferentiation of MSCs to ALCs. From the gene expression studies, it was established that the extract works almost in homogenous manner with other lipid inducers.     

Steroid sulfatase. Biosynthesis and processing in normal and mutant fibroblasts

Antibodies raised against steroid sulfatase purified from human placenta were used to follow the biosynthesis of this enzyme in human skin fibroblasts. Steroid sulfatase is synthesized as a membrane-bound Mr-63 500 polypeptide with asparagine-linked oligosaccharide chains. Within 2 days, newly synthesized steroid sulfatase is processed to a mature Mr-61 000 form. The decrease in size is due to processing of the oligosaccharide chains, which are cleavable by endoglucosaminidase H in both the early and the mature form of steroid sulfatase. The processing involves mannosidase(s) sensitive to 1-deoxy-manno-nojirimycin. The half-life of the steroid sulfatase polypeptides is 4 days. Synthesis of steroid-sulfatase-related polypeptides and steroid sulfatase activity were not detectable in fibroblasts from four patients with X-linked ichthyosis.     

Continuous Microbiological Transformation of Steroids

Continuous fermentation trials on the bioconversions of pregnadiene to pregnatriene by Septomyxa affinis and progesterone to 11α-hydroxyprogesterone by Rhizopus nigricans were conducted successfully in an eight-stage pilot plant reactor. The first stage was used as the mycelial growth stage while the steroid solutions were added continuously to stage 2, thus using the remaining stages as conversion vessels. Recoveries of 50 to 60% oxidized steroid (based on total steroid supplied) were obtained in both cases upon a contact time of 5 hr between mycelium and steroid. Longer contact times resulted in a gradual net loss of steroid. It was concluded that two-stage reactors (one growth stage and one conversion stage) were adequate for efficient continuous operation of such processes. The reaction volumes of both stages have to be kept in proper balance to insure optimal holdup times for both the cell growth and conversion steps.     

Reference intervals of urinary steroid metabolites using gas chromatography-mass spectrometry in Chinese adults

BACKGROUND: Urinary steroid profiling by GC or GC-MS are established clinical tools to complement other biochemical tests in the diagnosis and investigation of a wide range of adrenocortical disorders, but normative data on adults using the more specific GC-MS are lacking. Our objective was to set up the reference intervals of commonly detected urinary steroid metabolites as well as marker metabolites seen in disease states. METHOD: Apparently healthy adult Chinese males and females were recruited by completing health questionnaires. A 24-h urine specimen was collected from all the participants for urinary steroid profiling by GC-MS in cyclic scan mode. The analyzer was calibrated by using authentic steroid standards. Statistical methods recommended by the National Committee for Clinical Laboratory Standards were followed for setting up the reference intervals of various steroid metabolites. After outliers were excluded, the data were tested for the necessity to partition into sex-, menopausal status- and age-specific reference intervals. RESULTS: 83 males and 89 females were recruited for the study. Necessity to partition into sex-specific reference intervals was demonstrated for almost all steroid metabolites. Menopausal status and age also had a significant impact on steroid metabolite excretion, making separate reference intervals necessary. CONCLUSIONS: We have set up the normative data on the levels of urinary steroid metabolite excretion in Chinese adults for future reference in patient management and research in steroid metabolism.     

Administration of pregnenolone and dehydroepiandrosterone to guinea pigs and rats causes the accumulation of fatty acid esters of pregnenolone and dehydroepiandrosterone in plasma lipoproteins.

Steroids were administered continuously to guinea pigs and rats using subcutaneously applied silastic tubing implants, and the effects on circulating steroid and steroid conjugate levels were monitored. Using implants filled with pregnenolone, we observed that pregnenolone had a marked effect on increasing the levels of its fatty acid-esterified derivative, while dehydroepiandrosterone-releasing implants produced a rise in circulating nonconjugated dehydroepiandrosterone, androst-5-ene-3 beta,17 beta-diol, androstenedione, testosterone, and lipoidal derivatives of both dehydroepiandrosterone and androst-5-ene-3 beta,17 beta-diol. Implants filled with androstenedione produced a 20-fold increase in plasma androstenedione levels relative to untreated controls and a corresponding five-fold increase over control testosterone levels. No fatty acid-esterified derivative of testosterone could be detected within the plasma. Lipoproteins were isolated from both rats and guinea pigs treated with implants filled with pregnenolone or dehydroepiandrosterone. The steroid and steroid fatty acid esters present in each fraction were analyzed, revealing that approximately 75% of all the fatty acid esters of pregnenolone recovered in the lipoproteins was localized within the high-density lipoprotein (HDL) fraction of both guinea pig and rat plasma. Similarly, lipoidal dehydroepiandrosterone was found associated predominantly with the low-density lipoprotein and HDL fractions in the guinea pig, while in the rat this steroid conjugate was exclusively within the HDL fraction. High-density lipoprotein-incorporated tritiated pregnenolone fatty acid esters and dehydroepiandrosterone fatty acid esters were injected into castrated male guinea pigs to study the fate of these complexes.(ABSTRACT TRUNCATED AT 250 WORDS)