Gabriele Möller

The role of 17 beta-hydroxysteroid dehydrogenases.

The biological activity of steroid hormones is regulated at the pre-receptor level by several enzymes including 17 beta-hydroxysteroid dehydrogenases (17 beta -HSD). The latter are present in many microorganisms, invertebrates and vertebrates. Dysfunctions in human 17 beta-hydroxysteroid dehydrogenases result in disorders of biology of reproduction and neuronal diseases, the enzymes are also involved in the pathogenesis of various cancers. 17 beta-hydroxysteroid dehydrogenases reveal a remarkable multifunctionality being able to modulate concentrations not only of steroids but as well of fatty and bile acids. Current knowledge on genetics, biochemistry and medical implications is presented in this review.

Differences between Human Plasma and Serum Metabolite Profiles

Background Human plasma and serum are widely used matrices in clinical and biological studies. However, different collecting procedures and the coagulation cascade influence concentrations of both proteins and metabolites in these matrices. The effects on metabolite concentration profiles have not been fully characterized. Methodology/Principal Findings We analyzed the concentrations of 163 metabolites in plasma and serum samples collected simultaneously from 377 fasting individuals. To ensure data quality, 41 metabolites with low measurement stability were excluded from further analysis. In addition, plasma and corresponding serum samples from 83 individuals were re-measured in the same plates and mean correlation coefficients (r) of all metabolites between the duplicates were 0.83 and 0.80 in plasma and serum, respectively, indicating significantly better stability of plasma compared to serum (p = 0.01). Metabolite profiles from plasma and serum were clearly distinct with 104 metabolites showing significantly higher concentrations in serum. In particular, 9 metabolites showed relative concentration differences larger than 20%. Despite differences in absolute concentration between the two matrices, for most metabolites the overall correlation was high (mean r = 0.81±0.10), which reflects a proportional change in concentration. Furthermore, when two groups of individuals with different phenotypes were compared with each other using both matrices, more metabolites with significantly different concentrations could be identified in serum than in plasma. For example, when 51 type 2 diabetes (T2D) patients were compared with 326 non-T2D individuals, 15 more significantly different metabolites were found in serum, in addition to the 25 common to both matrices. Conclusions/Significance Our study shows that reproducibility was good in both plasma and serum, and better in plasma. Furthermore, as long as the same blood preparation procedure is used, either matrix should generate similar results in clinical and biological studies. The higher metabolite concentrations in serum, however, make it possible to provide more sensitive results in biomarker detection.

Phytoestrogens inhibit human 17β-hydroxysteroid dehydrogenase type 5

The 17beta-hydroxysteroid dehydrogenase type 5 (17beta-HSD 5) is involved in estrogen and androgen metabolism. In our study we tested the influence of environmental hormones, such as phytoestrogens (flavonoids, coumarins, coumestans), on reductive and oxidative 17beta-HSD activity of the human 17beta-hydroxysteroid dehydrogenase type 5 (17beta-HSD 5). These dietary substances were shown to be potent inhibitors of aromatase, different 17beta-HSDs and seem to play an important role in delay of development of hormone dependent cancers. Our studies show that reductive and oxidative activity of the enzyme are inhibited by many dietary compounds, especially zearalenone, coumestrol, quercetin and biochanin A. Among the group of flavones inhibitor potency is growing with increasing number of hydroxylations. We suggest that these substances are bound to the hydrophilic cofactor-binding pocket of the enzyme. An interesting inhibition pattern is observed for 18beta-glycyrrhetinic acid, which has no influence on the oxidative but only on the reductive reaction. This indicates that this substrate binds to pH- and cofactor-depending sites at the active center of the enzyme.

17β-Hydroxysteroid dehydrogenases (17β-HSDs) as therapeutic targets: protein structures, functions, and recent progress in inhibitor development.

17β-Hydroxysteroid dehydrogenases (17β-HSDs) are oxidoreductases, which play a key role in estrogen and androgen steroid metabolism by catalyzing final steps of the steroid biosynthesis. Up to now, 14 different subtypes have been identified in mammals, which catalyze NAD(P)H or NAD(P)(+) dependent reductions/oxidations at the 17-position of the steroid. Depending on their reductive or oxidative activities, they modulate the intracellular concentration of inactive and active steroids. As the genomic mechanism of steroid action involves binding to a steroid nuclear receptor, 17β-HSDs act like pre-receptor molecular switches. 17β-HSDs are thus key enzymes implicated in the different functions of the reproductive tissues in both males and females. The crucial role of estrogens and androgens in the genesis and development of hormone dependent diseases is well recognized. Considering the pivotal role of 17β-HSDs in steroid hormone modulation and their substrate specificity, these proteins are promising therapeutic targets for diseases like breast cancer, endometriosis, osteoporosis, and prostate cancer. The selective inhibition of the concerned enzymes might provide an effective treatment and a good alternative to the existing endocrine therapies. Herein, we give an overview of functional and structural aspects for the different 17β-HSDs. We focus on steroidal and non-steroidal inhibitors recently published for each subtype and report on existing animal models for the different 17β-HSDs and the respective diseases. Article from the Special issue on Targeted Inhibitors.

Determination of cDNA, gene structure and chromosomal localization of the novel human 17β‐hydroxysteroid dehydrogenase type 7

We have identified human 17β‐hydroxysteroid dehydrogenase type 7 (17β‐HSD 7). The novel human cDNA encodes a 37 kDa protein that shows 78 and 74% amino acid identity with rat and mouse 17β‐HSD 7, respectively. These enzymes are responsible for estradiol production in the corpus luteum during pregnancy, but are also present in placenta and several steroid target tissues (breast, testis and prostate) as revealed by RT‐PCR. The human 17β‐HSD 7 gene (HSD17B7) consists of nine exons and eight introns, spanning 21.8 kb and maps to chromosome 10p11.2 close to susceptibility loci for tumor progression, obesity and diabetes. The HSD17B7 promoter (1.2 kb) reveals binding sites for brain‐specific and lymphoid transcription factors corresponding to additional expression domains in hematopoietic tissues and the developing brain as identified by in silico Northern blot.

Discovery of phosphatidylcholines and sphingomyelins as biomarkers for ovarian endometriosis

BACKGROUND Current non-invasive diagnostic methods for endometriosis lack sensitivity and specificity. In search for new diagnostic biomarkers for ovarian endometriosis, we used a hypothesis-generating targeted metabolomics approach. METHODS In a case-control study, we collected plasma of study participants and analysed their metabolic profiles. We selected a group of 40 patients with ovarian endometriosis who underwent laparoscopic surgery and a control group of 52 healthy women who underwent sterilization at the University Clinical Centre Ljubljana, Slovenia. Over 140 targeted analytes included glycerophospholipids, sphingolipids and acylcarnitines. The analytes were quantified by electrospray ionization tandem mass spectrometry. For assessing the strength of association between the metabolite or metabolite ratios and the disease, we used crude and adjusted odds ratios. A stepwise logistic regression procedure was used for selecting the best combination of biomarkers. RESULTS Eight lipid metabolites were identified as endometriosis-associated biomarkers due to elevated levels in patients compared with controls. A model containing hydroxysphingomyelin SMOH C16:1 and the ratio between phosphatidylcholine PCaa C36:2 to ether-phospholipid PCae C34:2, adjusted for the effect of age and the BMI, resulted in a sensitivity of 90.0%, a specificity of 84.3% and a ratio of the positive likelihood ratio to the negative likelihood ratio of 48.3. CONCLUSIONS Our results suggest that endometriosis is associated with elevated levels of sphingomyelins and phosphatidylcholines, which might contribute to the suppression of apoptosis and affect lipid-associated signalling pathways. Our findings suggest novel potential routes for therapy by specifically blocking highly up-regulated isoforms of phosphpolipase A2 and lysophosphatidylcholine acyltransferase 4.

Molecular basis of D-bifunctional protein deficiency.

Peroxisomal disorders appear with a frequency of 1:5000 in newborns. They are caused either by peroxisomal assembly defects or by deficiencies of single peroxisomal enzymes. The phenotypes vary widely: affected humans may die very early in life within a few days to several months as a result of the impairment in essential peroxisomal functions as, for example, in Zellweger syndrome, or they may show only minor disabilities as is in acatalasemia. The deficiency of D-bifunctional protein, an enzyme involved in peroxisomal beta-oxidation of certain fatty acids and the synthesis of bile acids, causes a very severe, Zellweger-like phenotype. A number of different mutations in the gene coding for the enzyme were found in humans causing the total or partial loss of its enzymatic function. This paper gives a review of cases and their molecular basis.

17β-hydroxysteroid dehydrogenase type 7 — an ancient 3-ketosteroid reductase of cholesterogenesis

17beta-hydroxysteroid dehydrogenase type 7 (17beta-HSD7) is a novel estrogenic hydroxysteroid dehydrogenase from mammals. We modeled the three-dimensional structure of human 17beta-HSD7, analyzed the phylogeny of 17beta-HSD7 homologues and determined its expression pattern by in silico Northern blotting. Predominant expression is found not only in reproductive tissues (breast, ovary, placenta) but also in liver and developing brain, principal sites of cholesterol synthesis. The substrate binding pocket is opening towards a conserved membrane-associated helix, which is indicative for a conversion of a membrane component. 17beta-HSD7 shows significant homology to a yeast 3-ketosteroid reductase (ERG27) involved in ergosterol biosynthesis. Our results lead to the conclusion that 17beta-HSD7 is not only involved in estradiol production but plays another (and possibly more important) role as a 3-ketosteroid reductase in cholesterogenesis. This agrees with the striking absence of 17beta-HSD7 homologues in the complete genomes of Drosophila and C. elegans, which are both auxotrophic for cholesterol.

Targeted Metabolomics of Dried Blood Spot Extracts

Dried blood spot (DBS) samples are already successfully used in newborn screening and pharmacological analyses. The application of DBS matrix to further metabolomic methods will considerably extend the analytical options for the diagnostics of metabolic diseases. We present an MS/MS based method for the simultaneous extraction and quantification of 188 metabolites from dried blood spots. We provide a sensitive and reproducible method that adapts the AbsoluteIDQ™ p180 kit of Biocrates to the DBS matrix for the quantification of metabolites of different substance classes including amino acids, biogenic amines, free carnitine, acylcarnitines, hexoses, glycerophospholipids, lysophosphatidylcholines, phosphatidylcholines, and sphingolipids.

Recent advances in 17beta-hydroxysteroid dehydrogenases.

The metabolism of steroids at position 17 is catalysed by a growing number of 17beta-hydroxysteroid dehydrogenases (17beta-HSDs). Several human diseases like breast or prostate cancer, endometriosis,metabolic syndrome and mental diseases were associated with dysfunctions of 17beta-HSDs, which consequently became drug targets. This review will focus on identities of 17beta-HSDs and recent advances in analyses of their physiological roles in steroid and lipid metabolism. It will also address the potential of metabolomics in drug development.