Biology subjects

Storbeck, K.-H.

Publications and source records attributed to Storbeck, K.-H..

5 recordsLinked to original sources

bioRxiv2026

Optimisation and Validation of a UHPLC-MS/MS Assay Using Timed Post-Column Ammonium Fluoride Infusion for Sensitive Serum Steroids Quantitation

Steroid metabolomics has potential for identifying biomarkers and improving disease diagnosis. However, poor ionisation efficiency and low circulating concentrations of some steroids limit the broader clinical applications. Here, we developed a ultra-high-performance liquid chromatography - tandem mass spectrometry (UHPLC-MS/MS) assay for quantitation of 18 steroids in serum encompassing the mineralocorticoid, glucocorticoid and androgen biosynthesis pathways, including both classic and 11-oxygenated androgens. Steroid extraction from 100 L of serum was performed using supported liquid extraction with ethyl acetate elution. Ionisation efficiency of steroids was investigated using formic acid in the mobile phase and post-column infusion of ammonium fluoride. Ammonium fluoride enhanced the signal intensity of 3-keto-{Delta}4 steroids up to ninefold, however A-ring reduced steroids exhibited greater ionisation with formic acid. Thus, to optimise sensitivity, a timed post-column infusion of 2 mmol/L ammonium fluoride was employed during chromatographic separation. The lower limits of quantitation ranged from 0.04 to 2.1 nmol/L. Accuracy ranged from 82 to 113% and intra- and inter-assay imprecision was <10% and <13%, respectively. The assay was applied to analyse morning serum steroid concentrations in healthy individuals (55 women, 44 men; age-matched). Serum progesterone, 11-deoxycorticosterone, dehydroepiandrosterone and androstenedione were significantly higher in women; in contrast, testosterone and 5-dihydrotestosterone were higher in men. In summary, we have successfully developed and validated a UHPLC-MS/MS assay for multi-steroid profiling ad applied it to healthy individuals of both sexes.

biochemistry↗

bioRxiv2026

Inhibition of the androgen-activating enzyme AKR1C3 selectively decreases systemic and intra-adipose 11-oxygenated androgens in women

Androgen excess drives metabolic and reproductive complications in polycystic ovary syndrome (PCOS), affecting 10-15% of women globally. Aldo-keto reductase 1C3 (AKR1C3) converts inactive precursors from both the classic and the recently identified 11-oxygenated androgen pathways, generating testosterone and 11-ketotestosterone, respectively, which exert comparable androgen receptor activation. Both circulate in similar concentrations in premenopausal women while 11-ketotestosterone is predominant after menopause and in PCOS. Here, we show that adipocytes are a major site of AKR1C3 and androgen receptor expression, with increased expression in women and individuals with obesity. Using human female adipose tissue explants, we find a much higher activation of 11-oxygenated over classic androgens, observing a decrease in 11-oxygenated but not classic androgen activation by AKR1C3 inhibition. Correspondingly, we demonstrate that AKR1C3 inhibitor treatment in premenopausal women selectively disrupts the activation of 11-oxygenated androgens. Pharmacological targeting of AKR1C3 provides a novel strategy to alleviate systemic and intra-adipose 11-oxygenated androgen excess. One Sentence SummaryInhibition of the androgen-activating enzyme AKR1C3 results in a major decrease in 11-oxygenated but not classic androgens in women.

physiology↗

bioRxiv2023

Inhibition of the glucocorticoid-activating enzyme 11β-hydroxysteroid dehydrogenase type 1 drives concurrent 11-oxygenated androgen excess

Aldo-keto reductase 1C3 (AKR1C3) is a key enzyme in the activation of both classic and 11-oxygenated androgens. In adipose tissue, AKR1C3 is co-expressed with 11{beta}-hydroxysteroid dehydrogenase type 1 (HSD11B1), which catalyses the local activation of glucocorticoids but also the inactivation of 11-oxygenated androgens, and thus has the potential to counteract AKR1C3. Using a combination of in vitro assays and in silico modelling we show that HSD11B1 attenuates the biosynthesis of the potent 11-oxygenated androgen, 11-ketotestosterone, by AKR1C3. Employing ex vivo incubations of human female adipose tissue samples we show that inhibition of HSD11B1 results in the increased peripheral biosynthesis of 11-ketotestosterone. Moreover, circulating 11KT increased 2-3 fold in individuals with type 2 diabetes after receiving the selective oral HSD11B1 inhibitor AZD4017 for 35 days, thus confirming that HSD11B1 inhibition results in systemic increases in 11KT concentrations. Our findings show that HSD11B1 protects against excess 11KT production by adipose tissue, a finding of particular significance when considering the evidence for adverse metabolic effects of androgens in women. Therefore, when targeting glucocorticoid activation by HSD11B1 inhibitor treatment in women, the consequently increased generation of 11-ketotestosterone may offset beneficial effects of decreased glucocorticoid activation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=135 HEIGHT=200 SRC="FIGDIR/small/543687v2_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@f68f0forg.highwire.dtl.DTLVardef@12a5070org.highwire.dtl.DTLVardef@107231borg.highwire.dtl.DTLVardef@191ca58_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2020

Predominant activation of 11-oxygenated androgens by peripheral blood mononuclear cells

ContextAndrogens are important modulators of immune cell function impacting proliferation, differentiation and cytokine production. The local generation of active androgens from circulating androgen precursors is an important mediator of androgen action in peripheral androgen target cells or tissue. ObjectiveTo characterize the activation of classic and 11-oxygenated androgens in human peripheral blood mononuclear cells (PBMCs). MethodsPBMCs were isolated from healthy male donors and incubated ex vivo with precursors and active androgens of the classic and 11-oxygenated androgen pathways. Steroids were quantified by liquid chromatography-tandem mass spectrometry. The expression of genes encoding steroid-metabolizing enzymes was assessed by quantitative PCR. ResultsPBMCs generated 8-fold higher amounts of the active 11-oxygenated androgen 11-ketotestosterone than the classic androgen testosterone from their respective precursors. We identified the enzyme AKR1C3 as the major reductive 17{beta}-hydroxysteroid dehydrogenase in PBMCs responsible for both conversions and found that within the PBMC compartment natural killer cells are the major site of AKRC13 expression and activity. Steroid 5-reductase type 1 catalyzed the 5-reduction of classic but not 11-oxygenated androgens in PBMCs. Lag time prior to the separation of cellular components from whole blood increased 11KT serum concentrations in a time-dependent fashion, with significant increases detected from two hours after blood collection. Conclusions11-oxygenated androgens are the preferred substrates for androgen activation by AKR1C3 in PBMCs, primarily conveyed by natural killer cell AKR1C3 activity, yielding 11KT the major active androgen in PBMCs. Androgen metabolism by PBMCs can affect the measurement results of serum 11-ketotestosterone concentrations, if samples are not separated in a timely fashion.

biochemistry↗

bioRxiv2019

Bidirectional crosstalk between HIF and Glucocorticoid signalling in zebrafish larvae

In the last decades in vitro studies highlighted the potential for crosstalk between Hypoxia-Inducible Factor-(HIF) and glucocorticoid-(GC) signalling pathways. However, how this interplay precisely occurs in vivo is still debated. Here, we use zebrafish larvae (Danio rerio) to elucidate how and to what degree hypoxic signalling affects the endogenous glucocorticoid pathway and vice versa, in vivo. Firstly, our results demonstrate that in the presence of upregulated HIF signalling, both glucocorticoid receptor (Gr) responsiveness and endogenous cortisol levels are repressed in 5 days post fertilisation larvae. In addition, despite HIF activity being low at normoxia, our data show that it already impedes both glucocorticoid activity and levels. Secondly, we further analysed the in vivo contribution of glucocorticoids to HIF activity. Interestingly, our results show that both glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) play a key role in enhancing it. Finally, we found indications that glucocorticoids promote HIF signalling via multiple routes. Cumulatively, our findings allowed us to suggest a model for how this crosstalk occurs in vivo. Author summaryHypoxia is a common pathophysiological condition to which cells must rapidly respond in order to prevent metabolic shutdown and subsequent death. This is achieved via the activity of Hypoxia-Inducible Factors (HIFs), which are key oxygen sensors that mediate the ability of the cell to cope with decreased oxygen levels. Although it aims to restore tissue oxygenation and perfusion, it can sometimes be maladaptive and contributes to a variety of pathological conditions including inflammation, tissue ischemia, stroke and growth of solid tumours. In this regard, synthetic glucocorticoids which are analogous to naturally occurring steroid hormones, have been used for decades as anti-inflammatory drugs for treating pathological conditions which are linked to hypoxia (i.e. asthma, rheumatoid arthritis, ischemic injury). Indeed, previous in vitro studies highlighted the presence of a crosstalk between HIF and glucocorticoids. However, how this interplay precisely occurs in an organism and what the molecular mechanism is behind it are questions that still remain unanswered. Here, we provide a thorough in vivo genetic analysis, which allowed us to propose a logical model of interaction between glucocorticoid and HIF signalling. In addition, our results are important because they suggest a new route to downregulate HIF for clinical purposes.

physiology↗