Salivary cortisone as potential predictor of occupational exposure to noise and related stress.

Salivary cortisone strongly correlates with serum cortisol, and since it is less invasive to measure salivary cortisone than serum cortisol and easier than to measure cortisol in saliva, as its concentrations are much lower, we wanted to compare salivary cortisone and cortisol levels as markers of noise-induced stress reaction. The study included 104 participants aged 19-30 years, 50 of whom were exposed to occupational noise ≥85 dB(A) and 54 non-exposed, control students. All participants took samples of their saliva with Salivette® Cortisol synthetic swabs on three consecutive working days first thing in the morning. Salivary cortisone and cortisol levels were determined with high-performance liquid chromatography. In addition, they completed a 10-item Perceived Stress Scale (PSS-10) questionnaire, and occupationally noise-exposed participants also completed the Health and Safety Executive (HSE) questionnaire on occupational psychosocial risks. The exposed participants had significantly higher cortisone (P<0.001) and cortisol (P<0.001) levels than controls, and the correlation between cortisone and cortisol levels in the exposed participants was strong (ϱ =0.692, P<0.001), which suggests that salivary cortisone can replace cortisol measurements in saliva as a more reliable method than salivary cortisol and less invasive than serum cortisol. However, the level of perceived stress scored on PSS-10 in the exposed participants did not differ significantly from stress reported by controls, but correlated negatively with cortisone levels, which is contrary to our expectations and raises questions as to why.

DHRS7 (SDR34C1) - A new player in the regulation of androgen receptor function by inactivation of 5α-dihydrotestosterone?

DHRS7 (SDR34C1) has been associated with potential tumor suppressor effects in prostate cancer; however, its function remains largely unknown. Recent experiments using purified recombinant human DHRS7 suggested several potential substrates, including the steroids cortisone and Δ4-androstene-3,17-dione (androstenedione). However, the substrate and cofactor concentrations used in these experiments were very high and the physiological relevance of these observations needed to be further investigated. In the present study, recombinant human DHRS7 was expressed in intact HEK-293 cells in order to investigate whether glucocorticoids and androgens serve as substrates at sub-micromolar concentrations and at physiological cofactor concentrations. Furthermore, the membrane topology of DHRS7 was revisited using redox-sensitive green-fluorescent protein fusions in living cells. The results revealed that (1) cortisone is a substrate of DHRS7; however, it is not reduced to cortisol but to 20ß-dihydrocortisone, (2) androstenedione is not a relevant substrate of DHRS7, (3) DHRS7 catalyzes the oxoreduction of 5α-dihydrotestosterone (5αDHT) to 3α-androstanediol (3αAdiol), with a suppressive effect on androgen receptor (AR) transcriptional activity, and (4) DHRS7 is anchored in the endoplasmic reticulum membrane with a cytoplasmic orientation. Together, the results show that DHRS7 is a cytoplasmic oriented enzyme exhibiting 3α/20ß-hydroxysteroid dehydrogenase activity, with a possible role in the modulation of AR function. Further research needs to address the physiological relevance of DHRS7 in the inactivation of 5αDHT and AR regulation.

Variation of Human Milk Glucocorticoids over 24 hour Period.

Human milk (HM) contains a complex array of hormones, including members of the glucocorticoid family. The predominant glucocorticoids, cortisol and cortisone may influence the growth and behaviour of the breastfed infant. However, little is understood of the factors regulating the levels of these hormones within HM. The aim of the study was to examine HM cortisol and cortisone concentration, measured in samples collected at each feed during a 24 hour period. Twenty three exclusively breastfeeding mothers collected milk, prior to and after each breastfeeding session over 24 hour period at 3.2(1.60) months. HM cortisol and cortisone levels were measured using high pressure liquid chromatography mass spectroscopy. Cortisone was the predominant glucocorticoid (3.40 ng/ml), and cortisol was detected in all samples (1.62 ng/ml). A positive correlation was found between cortisone and cortisol (r = 0.61, y = 1.93 ± 0.24, p < 0.0001). Cortisol and cortisone concentrations were significantly higher in feeds in the morning (2.97 ng/ml and 4.88 ng/ml), compared to afternoon (1.20 ng/ml and 3.54 ng/ml), evening (0.69 ng/ml and 2.13 ng/ml) and night (1.59 and 3.27 ng/ml). No difference was found between glucocorticoids level of the milk expressed for collection either before or immediately after the breastfeed, or between milk collected from the left or right breast. This study shows that HM glucocorticoid concentrations exhibit a 24 hour pattern, with highest peak levels in the early morning, reflecting the circadian pattern as previously reported in plasma. Thus, HM glucocorticoid concentrations are likely to reflect those in the maternal circulation.

Chemometric evaluation of urinary steroid hormone levels as potential biomarkers of neuroendocrine tumors.

Neuroendocrine tumors (NETs) are uncommon tumors which can secrete specific hormone products such as peptides, biogenic amines and hormones. So far, the diagnosis of NETs has been difficult because most NET markers are not specific for a given tumor and none of the NET markers can be used to fulfil the criteria of high specificity and high sensitivity for the screening procedure. However, by combining the measurements of different NET markers, they become highly sensitive and specific diagnostic tests. The aim of the work was to identify whether urinary steroid hormones can be identified as potential new biomarkers of NETs, which could be used as prognostic and clinical course monitoring factors. Thus, a rapid and sensitive reversed-phase high-performance liquid chromatographic method (RP-HPLC) with UV detection has been developed for the determination of cortisol, cortisone, corticosterone, testosterone, epitestosterone and progesterone in human urine. The method has been validated for accuracy, precision, selectivity, linearity, recovery and stability. The limits of detection and quantification were 0.5 and 1 ng mL-1 for each steroid hormone, respectively. Linearity was confirmed within a range of 1-300 ng mL-1 with a correlation coefficient greater than 0.9995 for all analytes. The described method was successfully applied for the quantification of six endogenous steroid levels in human urine. Studies were performed on 20 healthy volunteers and 19 patients with NETs. Next, for better understanding of tumor biology in NETs and for checking whether steroid hormones can be used as potential biomarkers of NETs, a chemometric analysis of urinary steroid hormone levels in both data sets was performed.

Reactions of microsolvated organic compounds at ambient surfaces: droplet velocity, charge state, and solvent effects.

The exposure of charged microdroplets containing organic ions to solid-phase reagents at ambient surfaces results in heterogeneous ion/surface reactions. The electrosprayed droplets were driven pneumatically in ambient air and then electrically directed onto a surface coated with reagent. Using this reactive soft landing approach, acid-catalyzed Girard condensation was achieved at an ambient surface by directing droplets containing Girard T ions onto a dry keto-steroid. The charged droplet/surface reaction was much more efficient than the corresponding bulk solution-phase reaction performed on the same scale. The increase in product yield is ascribed to solvent evaporation, which causes moderate pH values in the starting droplet to reach extreme values and increases reagent concentrations. Comparisons are made with an experiment in which the droplets were pneumatically accelerated onto the ambient surface (reactive desorption electrospray ionization, DESI). The same reaction products were observed but differences in spatial distribution were seen associated with the "splash" of the high velocity DESI droplets. In a third type of experiment, the reactions of charged droplets with vapor phase reagents were examined by allowing electrosprayed droplets containing a reagent to intercept the headspace vapor of an analyte. Deposition onto a collector surface and mass analysis showed that samples in the vapor phase were captured by the electrospray droplets, and that instantaneous derivatization of the captured sample is possible in the open air. The systems examined under this condition included the derivatization of cortisone vapor with Girard T and that of 4-phenylpyridine N-oxide and 2-phenylacetophenone vapors with ethanolamine.

Expression profiles of human 11beta-hydroxysteroid dehydrogenases type 1 and type 2 in inflammatory bowel diseases.

Inflammatory bowel diseases such as Crohn's disease (CD) and ulcerative colitis (UC) are characterized by an increase in pro-inflammatory cytokines. On the other hand, endogenous cortisol is regarded as physiological compound to combat inflammation. The local activation of glucocorticoids is mediated by 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) which increases cortisol, and 11beta-HSD2 which decreases cortisol concentrations. We hypothesized that in inflamed tissues of patients suffering from inflammatory bowel diseases 11beta-HSD1 is upregulated whereas 11beta-HSD2 is downregulated. By using quantitative real-time PCR, we investigated the transcription levels of 11beta-HSD1 and 11beta-HSD2 in patients diagnosed with CD or UC. Expression of 11beta-HSD1 was significantly elevated in inflamed tissue compared to non-inflamed colonic tissue in both, CD (2.7-fold) and UC (3.8-fold), whereas 11beta-HSD2 expression was decreased in the same samples. In both diseases, male patients showed a more pronounced upregulation of 11beta-HSD1 (CD: 4.8-fold, UC: 6.5-fold) compared to females (CD: 1.8-fold, UC: 1.8-fold), a fact which might be due to the higher levels of circulating anti-inflammatory estrogens in women. Our data support the hypothesis that both enzymes play a crucial role in inflammation by affecting local tissue ratios between active and inactive glucocorticoids.

Circulating 11beta-hydroxysteroid dehydrogenase type 1 mRNA and cardiovascular risk factors.

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) has been implicated in the pathogenesis of central obesity and metabolic syndrome. It has been shown that tissue-specific mRNA is present in blood, raising the possibility that quantifiable levels of 11beta-HSD1 mRNA may also be present in circulation. The aim of this study was to determine whether a relationship exists between circulating 11beta-HSD1 mRNA levels and various cardiovascular risk factors. Whole blood was collected using PAXgene Blood RNA tubes from 73 subjects. Total RNA was extracted, reverse-transcribed to cDNA, and 11beta-HSD1 mRNA was measured by quantitative real time PCR. The median BMI was 24 (range = 17-36) and the concentration of 11beta-HSD1 mRNA tended to decrease with increasing BMI (r=-0.206; P= 0.08). Multiple linear regression analysis identified BMI as an independent predictor of mRNA levels (beta=-0.412, P= 0.002). Other factors included in the analysis are age (beta= 0.952, P < 0.001), smoking (beta=-0.228, P= 0.027), alcohol intake (beta= 0.479, P < 0.001), and duration of menopause (beta=-1.602, P < 0.001). The results suggest a net decrease in systemic expression of 11beta-HSD1 with increased smoking, BMI, and duration of menopause. Increased systemic expression is seen with increasing age and alcohol intake.

Crystal structure of human liver Delta4-3-ketosteroid 5beta-reductase (AKR1D1) and implications for substrate binding and catalysis.

AKR1D1 (steroid 5beta-reductase) reduces all Delta(4)-3-ketosteroids to form 5beta-dihydrosteroids, a first step in the clearance of steroid hormones and an essential step in the synthesis of all bile acids. The reduction of the carbon-carbon double bond in an alpha,beta-unsaturated ketone by 5beta-reductase is a unique reaction in steroid enzymology because hydride transfer from NADPH to the beta-face of a Delta(4)-3-ketosteroid yields a cis-A/B-ring configuration with an approximately 90 degrees bend in steroid structure. Here, we report the first x-ray crystal structure of a mammalian steroid hormone carbon-carbon double bond reductase, human Delta(4)-3-ketosteroid 5beta-reductase (AKR1D1), and its complexes with intact substrates. We have determined the structures of AKR1D1 complexes with NADP(+) at 1.79- and 1.35-A resolution (HEPES bound in the active site), NADP(+) and cortisone at 1.90-A resolution, NADP(+) and progesterone at 2.03-A resolution, and NADP(+) and testosterone at 1.62-A resolution. Complexes with cortisone and progesterone reveal productive substrate binding orientations based on the proximity of each steroid carbon-carbon double bond to the re-face of the nicotinamide ring of NADP(+). This orientation would permit 4-pro-(R)-hydride transfer from NADPH. Each steroid carbonyl accepts hydrogen bonds from catalytic residues Tyr(58) and Glu(120). The Y58F and E120A mutants are devoid of activity, supporting a role for this dyad in the catalytic mechanism. Intriguingly, testosterone binds nonproductively, thereby rationalizing the substrate inhibition observed with this particular steroid. The locations of disease-linked mutations thought to be responsible for bile acid deficiency are also revealed.

Carbonyl reductases and pluripotent hydroxysteroid dehydrogenases of the short-chain dehydrogenase/reductase superfamily.

Carbonyl reduction of aldehydes, ketones, and quinones to their corresponding hydroxy derivatives plays an important role in the phase I metabolism of many endogenous (biogenic aldehydes, steroids, prostaglandins, reactive lipid peroxidation products) and xenobiotic (pharmacologic drugs, carcinogens, toxicants) compounds. Carbonyl-reducing enzymes are grouped into two large protein superfamilies: the aldo-keto reductases (AKR) and the short-chain dehydrogenases/reductases (SDR). Whereas aldehyde reductase and aldose reductase are AKRs, several forms of carbonyl reductase belong to the SDRs. In addition, there exist a variety of pluripotent hydroxysteroid dehydrogenases (HSDs) of both superfamilies that specifically catalyze the oxidoreduction at different positions of the steroid nucleus and also catalyze, rather nonspecifically, the reductive metabolism of a great number of nonsteroidal carbonyl compounds. The present review summarizes recent findings on carbonyl reductases and pluripotent HSDs of the SDR protein superfamily.

Conformational flexibility in crystal structures of human 11beta-hydroxysteroid dehydrogenase type I provide insights into glucocorticoid interconversion and enzyme regulation.

Human 11beta-hydroxysteroid dehydrogenase type I (11beta-HSD1) is an ER-localized membrane protein that catalyzes the interconversion of cortisone and cortisol. In adipose tissue, excessive cortisol production through 11beta-HSD1 activity has been implicated in the pathogenesis of type II diabetes and obesity. We report here biophysical, kinetic, mutagenesis, and structural data on two ternary complexes of 11beta-HSD1. The combined results reveal flexible active site interactions relevant to glucocorticoid recognition and demonstrate how four 11beta-HSD1 C termini converge to form an as yet uncharacterized tetramerization motif. A C-terminal Pro-Cys motif is localized at the center of the tetramer and forms reversible enzyme disulfides that alter enzyme activity. Conformational flexibility at the tetramerization interface is coupled to structural changes at the enzyme active site suggesting how the central Pro-Cys motif may regulate enzyme activity. Together, the crystallographic and biophysical data provide a structural framework for understanding 11beta-HSD1 activities and will ultimately facilitate the development of specific inhibitors.

Aqueous chromatography utilizing pH-/temperature-responsive polymer stationary phases to separate ionic bioactive compounds.

Cross-linked poly(N-isopropylacrylamide-co-acrylic acid) (poly(IPAAm-co-AAc))-grafted silica bead surfaces were prepared and applied as new column matrix materials that exploit temperature-responsive anionic chromatography to separate basic bioactive compounds, specifically catecholamine derivatives, in aqueous mobile phases. Since poly(IPAAm-co-AAc) has a well-known temperature-responsive phase transition and apparent pKa shift, polymer-grafted silica bead surfaces are expected to exhibit simultaneous hydrophilic/hydrophobic and charge density alterations under thermal stimuli. Elution behavior of catecholamine derivatives from a copolymer-modified bead packed column was monitored using aqueous mobile-phase HPLC under varying temperature and pH. Catecholamine derivatives had higher retention times on poly(IPAAm-co-AAc) columns at higher pH in comparison with those on noncharged PIPAAm reference columns, suggesting an electrostatic interaction as a separation mode. Temperature also affected the retention behavior of catecholamine derivatives. Optimal separation of four catecholamine derivatives was achieved at elevated temperature, 50 degrees C, and at pH 7.0. This is due to the increased hydrophobicity of the stationary phase as evidenced by the elution of a nonionic hydrophobic steroid. From these results, mutual influences of both electrostatic and hydrophobic interactions between basic catecholamine derivatives and pH-/temperature-responsive surfaces are noted. Consequently, elution of weakly charged bioactive compounds is readily regulated through the modulation of stationary-phase thermoresponsive hydrophilic/hydrophobic and charge density changes.

The effect of structural QSAR parameters on skin penetration.

The permeability coefficients (log kp) of solutes through stratum corneum have been previously related to the octanol-water partition coefficients (log Poct) and solvatochromic parameters. In this study, permeation coefficient data are related to the theoretical chemistry-derived structural parameters and also molecular connectivity and molecular shape indices. The results show that these parameters are comparable with the solvatochromic parameters in correlation with log kp. Log Poct can be corrected by the theoretical parameters to explain permeation coefficients and the equilibrium distribution of compounds between the stratum corneum and water (log Km). Diffusion estimated from the expression log(D/h)=log kp-log Km, where D is the diffusion coefficient and h is the path length for diffusion was also analyzed successfully by the structural parameters.

Detection of exogenous intake of natural corticosteroids by gas chromatography/combustion/isotope ratio mass spectrometry: application to misuse in sport.

A detailed procedure for the analysis of exogenous hydrocortisone and cortisone in urine by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) is proposed. As urinary levels of hydrocortisone are rather low for GC/C/IRMS analysis, the focus is on the main corticosteroid metabolites, tetrahydrocortisone (THE) and tetrahydrocortisol (THF). Following different solid phase extraction purifications, THE and THF are oxidized to 5beta-androstanetrione before analysis by GC/C/IRMS. Significant differences in delta(13)C per thousand values of synthetic natural corticosteroids and endogenous human corticosteroids have been observed. Therefore, a positive criterion, to detect exogenous administration of synthetic corticosteroids in anti-doping control, is proposed.

HINT: a new method of empirical hydrophobic field calculation for CoMFA.

An empirical hydrophobic field-like 3D function has been calculated with the program HINT (hydrophobic interactions) and imported into the SYBYL implementation of CoMFA (Comparative Molecular Field Analysis). The addition of hydrophobicity appears to offer increased chemical interpretability of CoMFA models. An example is given using the steroid model reported by Cramer et al. (J. Am. Chem. Soc., 110 (1988) 5959). While addition of the HINT field did not improve statistical parameters in this model, the CoMFA coefficient contours from the hydrophobic field unambiguously define the most active steroid molecules in the chemical terms of hydrophobic and polar substituents.

Beta-cyclodextrin/steroid complexation: effect of steroid structure on association equilibria.

Molecular associations of beta-cyclodextrin (beta-CyD) with four steroids (cortisone, hydrocortisone, progesterone, and testosterone) have been studied using phase-solubility and spectroscopic techniques. Phase solubility diagrams could be categorized as B type. The complexes are formed at the stoichiometric ratios of 1:2 (drug:beta-CyD). A mathematical model has been proposed to calculate the apparent stability constants. The results suggest that the inclusion of a steroid molecule into the first beta-CyD cavity is thermodynamically more favorable over the association of 1:1 complex with the second beta-CyD molecule except for cortisone, which exhibits anomalous behavior. A mechanism of complexation has been proposed based on the apparent stability constants and chemical structures of the steroids and beta-CyD. It suggests that complexation is first brought about by inclusion of the five-member cyclopentane ring of the steroid molecule into the first beta-CyD cavity. The 1:1 complex subsequently binds with the second beta-CyD to form the 1:2 complex. The association constants of steroid/beta-CyD complexes are of the following order: progesterone greater than cortisone greater than testosterone greater than hydrocortisone. The order of aqueous solubilities of the complexes is hydrocortisone greater than cortisone greater than testosterone greater than progesterone.