Influence of labor on direct and indirect determinants of placental 11beta-hydroxysteroid dehydrogenase activity.

PURPOSE: Labor is a complex process involving multiple para-, auto- and endocrine cascades. The interaction of cortisol, corticotropin-releasing hormone (CRH) and progesterone is essential. The action of cortisol on the human feto-placental unit is regulated by 11beta-hydroxysteroid dehydrogenase type 2 (11ß-HSD2/HSD11B2) that converts cortisol into inactive cortisone. The majority of studies on the assessment of placental 11ß-HSD2 function determined indirect activity parameters. It remains elusive if indirect measurements correlate with enzymatic function and if these parameters are affected by potential confounders (e.g., mode of delivery). Thus, we compared determinants of indirect 11ß-HSD2 tissue activity with its direct enzymatic turnover rate in placental samples from spontaneous births and cesarean (C)-sections. METHODS: Using LC-MS/MS, we determined CRH, cortisol, cortisone, progesterone and 17-hydroxy(OH)-progesterone in human term placentas (spontaneous birth vs. C-section, n = 5 each) and measured the enzymatic glucocorticoid conversion rates in placental microsomes. Expression of HSD11B1, 2 and CRH was determined via qRT-PCR in the same samples. RESULTS: Cortisol-cortisone ratio correlated with direct microsomal enzymatic turnover. While this observation seemed independent of sampling site, a strong influence of mode of delivery on tissue steroids was observed. The mRNA expression of HSD11B2 correlated with indirect and direct cortisol turnover rates in C-section placentas only. In contrast to C-sections, CRH, cortisol and cortisone levels were significantly increased in placental samples following spontaneous birth. CONCLUSION: Labor involves a series of complex hormonal processes including activation of placental CRH and glucocorticoid metabolism. This has to be taken into account when selecting human cohorts for comparative analysis of placental steroids.

Adhesive blood microsampling systems for steroid measurement via LC-MS/MS in the rat.

INTRODUCTION: Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) allows for the direct analysis of multiple hormones in a single probe with minimal sample volume. Rodent-based animal studies strongly rely on microsampling, such as the dry blood spot (DBS) method. However, DBS suffers the drawback of hematocrit-dependence (non-volumetric). Hence, novel volumetric microsampling techniques were introduced recently, allowing sampling of fixed accurate volumes. We compared these methods for steroid analysis in the rat to improve inter-system comparability. EXPERIMENTAL: We analyzed steroid levels in blood using the absorptive microsampling devices Whatman® 903 Protein Saver Cards, Noviplex™ Plasma Prep Cards and the Mitra™ Microsampling device and compared the obtained results to the respective EDTA plasma levels. Quantitative steroid analysis was performed via LC-MS/MS. For the determination of the plasma volume factor for each steroid, their levels in pooled blood samples from each human adults and rats (18weeks) were compared and the transferability of these factors was evaluated in a new set of juvenile (21days) and adult (18weeks) rats. Hematocrit was determined concomitantly. RESULTS: Using these approaches, we were unable to apply one single volume factor for each steroid. Instead, plasma volume factors had to be adjusted for the recovery rate of each steroid and device individually. The tested microsampling systems did not allow the use of one single volume factor for adult and juvenile rats based on an unexpectedly strong hematocrit-dependency and other steroid specific (pre-analytic) factors. DISCUSSION: Our study provides correction factors for LC-MS/MS steroid analysis of volumetric and non-volumetric microsampling systems in comparison to plasma. It argues for thorough analysis of chromatographic effects before the use of novel volumetric systems for steroid analysis.

Species differences of 11beta-hydroxysteroid dehydrogenase type 2 function in human and rat term placenta determined via LC-MS/MS.

INTRODUCTION: Glucocorticoid-induced fetal programming has been associated with negative metabolic and cardiovascular sequelae in the adult. The placental enzyme 11beta-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) shields the fetus from maternal glucocorticoid excess by catalyzing the conversion of these hormones into biologically inactive derivatives. In vivo experiments addressing placental barrier function are mostly conducted in rodents. Therefore we set out to characterize species-specific differences of rat and human placental 11ß-HSD2 steroid turnover, introducing Liquid Chromatography Tandem Mass-Spectrometry (LC-MS/MS) as a tool for rat tissue analysis. MATERIALS AND METHODS: Using LC-MS/MS we determined corticotropin-releasing hormone (CRH), cortisol (F), cortisone (E), corticosterone (B) and 11-dehydrocorticosterone (A) in human and rat placenta at term and measured the enzymatic 11ß-HSD glucocorticoid conversion-rates in placental microsomes of both species. In parallel, further glucocorticoid derivatives and sex steroids were determined in the same placental samples. RESULTS: In contrast to the human placenta, we did not detect CRH in the rat placenta. While cortisol (F) and cortisone (E) were exclusively present in human term placenta (E/F-ratio >1), rat placenta showed significant levels of corticosterone (B) and 11-dehydrocorticosterone (A), with an A/B-ratio <1. In line with these species-specific findings, human placenta showed a prominent 11ß-HSD2 activity, while in rat placenta higher 11ß-HSD1 glucocorticoid turnover rates were determined. DISCUSSION: Placental steroid metabolism of human and rat shows relevant species-specific differences, especially regarding the barrier function of 11ß-HSD2 at term. The exclusive expression of CRH in the human placenta further points to relevant differences in the regulation of parturition in rats. Consideration of these findings is warranted when transferring results from rodent placental glucocorticoid metabolism into humans.

Measurement of amniotic fluid steroids of midgestation via LC-MS/MS.

INTRODUCTION: Analysis of steroids by mass spectrometry (MS) has evolved into a reliable tool for the simultaneous detection of multiple steroids. As amniotic fluid (AF) and fetal serum composition of early pregnancy are closely related, the analysis of AF can yield information on the physiological status of the developing fetus. We evaluated the use of liquid-chromatography tandem mass spectrometry (LC-MS/MS) for AF steroid analysis, including the analysis of its sensitivity and accuracy for gender verification in healthy subjects. MATERIALS AND METHODS: AF of 78 male and 94 female healthy newborns was analyzed by LC-MS/MS at 16 weeks of gestation. The levels of androstenedione, corticosterone, cortisol, cortisone, deoxycorticosterone, 11-deoxycortisol, dehydroepiandrosterone (DHEA), dehydroepiandrosterone-sulfate (DHEA-S), 17-hydroxyprogesterone, progesterone (17-OHP) and testosterone were measured. Steroid levels were compared to RIA and GC-MS levels of midgestation from the literature. Cross-validated logistic regression was used to obtain statistical predictions of gender at birth from testosterone and the above steroids. RESULTS: LC-MS/MS analysis of AF steroids yielded comparable results with published GC-MS data. Gender specific differences were found for androstenedione and testosterone concentrations with higher levels in the male fetus. In contrast to published RIA data no gender specific differences were observed for 17-hydroxyprogesterone and dehydroepiandrosterone AF concentrations. Testosterone concentrations yielded highly accurate predictions for male gender at birth. Additional analysis of further steroids did neither increase the accuracy, sensitivity nor specificity of this prediction. The estimated optimal cut-off value for amniotic testosterone level was 0.074 µg/L for healthy male newborns. CONCLUSIONS: LC-MS/MS is a reliable method for the determination of steroids in amniotic fluid. The determination of testosterone in amniotic fluid by LC-MS/MS in early pregnancy of healthy subjects can be used to offer a reliable prediction of fetal gender at birth.

Cluster-controlled dimerisation in supramolecular ruthenium photosensitizer-polyoxometalate systems.

A supramolecular reaction system is reported where a labile molecular metal oxide cluster enables the unprecedented dimerisation of ruthenium photosensitizers [Ru(L)2(tmbiH2)](2+) (L = 4,4'-di-tert-butyl-2,2'-bipyridine (1a) or 2,2'-bipyridine (1b); tmbiH2 = 5,5',6,6'-tetramethyl-2,2'-bibenzimidazole). In the presence of [Mo8O26](4-) clusters (2) the dimerisation is triggered by the in situ conversion of [Mo8O26](4-) to [Mo6O19](2-) which results in the release of hydroxide ions. Simultaneous deprotonation of the pH-sensitive tmbiH2-ligands starts the dimerisation, resulting in the formation of the dinuclear complex [(Ru(L)2)2(tmbi)](2+) (L = 4,4'-di-tert-butyl-2,2'-bipyridine (3) or 2,2'-bipyridine (4)). The dimerisation reaction can be suppressed when 2 is replaced by a stable polyoxomolybdate cluster, [Mo5O15(PhPO3)2](4-) (5) and the reaction between 1a and 5 leads to the formation of hydrogen-bonded supramolecular aggregates 6. The solution and solid-state interactions in these systems were investigated using a range of spectroscopic and crystallographic techniques and compounds 3, 4 and 6 were characterized using single-crystal XRD.

Solution and solid-state interactions in a supramolecular ruthenium photosensitizer-polyoxometalate aggregate.

The intermolecular interactions between a ruthenium-based photosensitizer ([Ru(tbbpy)(2)(biH(2))](2+)) and a molecular metal oxide ([ß-Mo(8)O(26)](4-)) are investigated in solution and in the solid state. The supramolecular interactions were studied using (1)H-NMR, UV-Vis and emission spectroscopy, ESI mass spectrometry and single-crystal X-ray diffraction. The formation of supramolecular aggregates was observed both in the crystal lattice and in solution. In addition, it is shown that aggregation in solution can be controlled by the competitive formation of ion pairs.