Steroid analysis by pH-mediated stacking MEKC

This dissertation is based on research that led to the development and application of capillary electrophoresis method for the analysis of sex steroid hormones in the blood or plasma of fish species. The analysis of steroid hormones is essential to reveal chemical compounds that are suspected in endocrine disruption, but it is challenging due to the similarity of the chemical structures of steroids, their low concentrations, and limited volumes of plasma or blood samples in fish available for analysis. There is therefore an acute need to develop reliable accurate and systematic analytical methods applicable for the analysis of structurally similar steroid hormones at very low concentrations. The method developed here is based on micellar electrokinetic chromatography, a type of capillary electrophoresis that incorporates secondary equilibria. The method utilizes pH-stacking for steroid preconcentration to improve the detection limits. This method of pH-stacking is accomplished by using charged derivatives of cyclodextrin which become neutral (protonated) or anionic (deprotonated) based on the pH of the sample buffer. Preconcentration by means of pH-stacking occurs upon introduction of the sample into the capillary at the pH junction, resulting in a fast and efficient separation analysis of steroids. Using the developed method the separation of eight targeted steroids, that include alpha,beta-dihydroxyprogesterone, ethynylestradiol, 17beta-estradiol, estrone, hydroxyprogesterone, 11-ketotestosterone, progesterone, and testosterone is achieved in less than 4 min which is substantially faster than steroid analysis by immunoassay, GC-MS or LC-MS. For all targeted steroids, the within-day and day-to-day reproducibility in migration time is \u3c1 and \u3c2% relative standard deviation (RSD), respectively. The reproducibility in peak area obtained in aqueous samples is below 6% and 22% (RSD) within-day and day-to-day respectively. The limits of detection range from 2 to 14 nM using a 60 s electrokinetic injection. The method is validated by measuring the recovery of standard steroids added to the aqueous or fish plasma samples prior to sample preparation. The recovery of testosterone and 17beta-estradiol added to the fish plasma prior to sample preparation range from 74% to 102%. The method is successfully applied to the determination of sex steroid levels in blood plasma of yellow perch captured from natural aqueous habitats. The results are compared to radioimmunoassay

Biotransformation of Steroids Using Different Microorganisms

The introduction of a hydroxyl group “biohydroxylation” in the steroid skeleton is an important step in the synthesis of new steroids used physiologically as hormones and active drugs. There are currently about 300 known steroid drugs whose production constitutes the second category within the pharmaceutical market after antibiotics. Several biotransformations at industrial scale have been applied in the production of steroid hormones and drugs, which have functionalized different types of raw materials by means of chemo-, regio-, and stereoselective reactions (hydroxylation, Baeyer-Villiger oxidation, oxidation reactions, reduction of group carbonyl, isomerization, and Michael additions, condensation reactions, among others). In Green Chemistry, biotransformations are an important chemical methodology toward more sustainable industrial processes

The steroid metabolome in women with premenstrual dysphoric disorder during GnRH agonist-induced ovarian suppression: effects of estradiol and progesterone addback

Clinical evidence suggests that symptoms in premenstrual dysphoric disorder (PMDD) reflect abnormal responsivity to ovarian steroids. This differential steroid sensitivity could be underpinned by abnormal processing of the steroid signal. We used a pharmacometabolomics approach in women with prospectively confirmed PMDD (n=15) and controls without menstrual cycle-related affective symptoms (n=15). All were medication-free with normal menstrual cycle lengths. Notably, women with PMDD were required to show hormone sensitivity in an ovarian suppression protocol. Ovarian suppression was induced for 6 months with gonadotropin-releasing hormone (GnRH)-agonist (Lupron); after 3 months all were randomized to 4 weeks of estradiol (E2) or progesterone (P4). After a 2-week washout, a crossover was performed. Liquid chromatography/tandem mass spectrometry measured 49 steroid metabolites in serum. Values were excluded if >40% were below the limit of detectability (n=21). Analyses were performed with Wilcoxon rank-sum tests using false-discovery rate (q<0.2) for multiple comparisons. PMDD and controls had similar basal levels of metabolites during Lupron and P4-derived neurosteroids during Lupron or E2/P4 conditions. Both groups had significant increases in several steroid metabolites compared with the Lupron alone condition after treatment with E2 (that is, estrone-SO4 (q=0.039 and q=0.002, respectively) and estradiol-3-SO4 (q=0.166 and q=0.001, respectively)) and after treatment with P4 (that is, allopregnanolone (q=0.001 for both PMDD and controls), pregnanediol (q=0.077 and q=0.030, respectively) and cortexone (q=0.118 and q=0.157, respectively). Only sulfated steroid metabolites showed significant diagnosis-related differences. During Lupron plus E2 treatment, women with PMDD had a significantly attenuated increase in E2-3-sulfate (q=0.035) compared with control women, and during Lupron plus P4 treatment a decrease in DHEA-sulfate (q=0.07) compared with an increase in controls. Significant effects of E2 addback compared with Lupron were observed in women with PMDD who had significant decreases in DHEA-sulfate (q=0.065) and pregnenolone sulfate (q=0.076), whereas controls had nonsignificant increases (however, these differences did not meet statistical significance for a between diagnosis effect). Alterations of sulfotransferase activity could contribute to the differential steroid sensitivity in PMDD. Importantly, no differences in the formation of P4-derived neurosteroids were observed in this otherwise highly selected sample of women studied under controlled hormone exposures

Microorganisms as Biocatalysts and Enzyme Sources

Microbial-catalyzed biotransformations have considerable potential for the generation of an enormous variety of structurally diversified organic compounds, especially natural products with complex structures like triterpenoids, flavonoids, steroids, steroidal saponins, and sesquiterpenoids. They offer efficient and economical ways to produce semisynthetic analogues and novel lead molecules. Microorganisms such as bacteria and fungi could catalyze chemo-, regio-, and stereospecific hydroxylations of diverse substrates that are extremely difficult to produce by chemical routes. During recent years, considerable research has been performed on the microbial transformation of bioactive compounds, in order to obtain biologically active molecules with diverse structural features. In green chemistry, biotransformations are an important chemical methodology toward more sustainable industrial processes

The Progesterone Hydroxylase Cytochrome P450 Multicomponent System of Streptomyces roseochromogenes: Purification, Characterisation and Regulation

PhDStreptomyces roseochromogenes, NCIB 10984, hydroxylates exogenous progesterone to 16a hydroxyprogesterone and thereafter in a second phase bioconversion to 2ß, 16a-dihydroxyprogesterone. Characterisation of this reaction was carried out at the whole cell level. The cellular components responsible for this reaction were also purified to homogeneity. S. roseochromogenes contains a cytochrome P450 and two electron transfer proteins, roseoredoxin and roseoredoxin reductase. A reconstituted incubation containing these purified proteins and the natural electron donor, NADH. produced identical hydroxyprogesterone metabolites as intact cells. In sodium periodate (Na104) supported incubations, the initial rate of progesterone hydroxylation was marginally higher than in the natural reconstituted system but the product yield was significantly lower. The yield data showed that the reconstituted natural pathway, supported multiple rounds of hydroxylation in contrast to a likely single round by a minority of P450s in the periodate reaction. When S. roseochromogenes was incubated with exogenous progesterone for 25 h the major metabolite, 16a-hydroxyprogesterone was produced in 3.6 fold excess to the minor metabolite 2ß, 16a-dihydroxyprogesterone. In a reconstituted system containing highly purified progesterone 16a-hydroxylase cytochrome P450, roseoredoxin and roseoredoxin reductase, both metabolites were produced but in a 10: 1 ratio. When S. roseochromogenes was preincubated with progesterone and the purified components of the hydroxylase system assayed as before, the ratio of 16a-hydroxyprogesterone to 2ß, 16adihydroxyprogesterone produced, decreased to 2.8: 1, virtually identical to the ratio in whole cell biotransformations. Reconstitution assays containing all combinations of hydroxylase proteins purified from progesterone preincubated and control cells, identified roseoredoxin as solely responsible for the observed changes in in vitro metabolite ratios. The fact that the 2.8: 1 ratio was also obtained when S. roseochromogenes was exposed to cycloheximide prior to progesterone pre-incubation; pointed to post translation modification of roseoredoxin. Separation of two isoforms by 2-D isoelectric focusing supported this proposition. A partial 10 amino acid sequence was obtained for both the cytochrome P450 and roseoredoxin for the purpose of probe design for eventual cloning. An amino acid sequence search revealed this P450 to be unique and unlike any other known P450 sequence. These two proteins were also successfully crystallised by hanging drop vapour diffusion trials, giving isomorphous crystals. These crystals will be used for structure determinations pending further growth

Tilapia male urinary pheromone stimulates female reproductive axis

Mozambique tilapia males congregate in leks where they establish dominance hierarchies and attract females to spawn in sandy pits. Dominant males store more urine than subordinates and the pattern of urination and the high sensitivity of females to male urine suggest chemical signalling via the urine. Here we show that pre-ovulated and post-spawn females when exposed to dominant male urine increased significantly, in less than 1 h, the release rate of the maturation-inducing steroid 17,20bdihydroxypregn- 4-en-3-one which is maintained elevated for at least 6 h. This indicates a pheromonal role for male urine in the synchronisation of spawning. Furthermore, we show that the lack of affinity of 17,20bP to sex steroid binding globulin explains, at least partly, its rapid release and lack of detection in the blood. Thus tilapia urine involvement in several communication processes confirms that cichlids have evolved a sophisticated chemical signalling system together with their complex visual, acoustic and behavioural displays

Non-terpenoid biotransformations by Mucor species

Biotransformation is an important tool for the structural modification of organic compounds, especially natural products with complex structures, which are difficult to achieve using ordinary methods. It is also useful as a model for mammalian metabolism due to similarities between mammalian and microbial enzyme systems. The development of novel biocatalytic methods is a continuously growing area of chemistry, microbiology, and genetic engineering, and novel microorganisms and/or their enzymes are being screened intensively. This review covers the transformation of non-terpenoid compounds such as steroids, coumarins, flavonoids, drugs, pesticides and others by Mucor spp. up to the end of 2012

Efectividad en amenaza de aborto de progesterona natural micronizada comparado con caproato 17 dihidroxiprogesterona

Objetivo: Comparar la terapia con progesterona natural micronizada respecto a caproato 17 dihidroxiprogesterona en el manejo de amenaza de aborto. Material y métodos: Se llevó a cabo un estudio de cohorte histórica. Se incluyeron a 120 pacientes con amenaza de aborto, los cuales se dividieron en 2 grupos: pacientes expuestas a progesterona micronizada y pacientes expuestas a caproato 17 dihidroxiprogesterona; aplicándose el riesgo relativo, y la prueba estadística chi cuadrado. Resultados La frecuencia de abordo en pacientes expuestas a progesterona natural micronizada fue 22%. La frecuencia de aborto en pacientes expuestas a caproato 17 dihidroxiprogesterona fue 42%. La efectividad de progesterona natural micronizada es superior a la de caproato 17 dihidroxiprogesterona en el manejo de amenaza de aborto con un RR= 1.34 (p<0.05). Conclusión: La terapia con progesterona natural micronizada es superior a caproato 17 dihidroxiprogesterona en el manejo de amenaza de aborto.Objective: to compare the therapy with micronized natural progesterone with caproate 17 dihydroxyprogesterone in the management of threatened abortion. Material and methods: A historical cohort study.120 patients with threatened abortion were included, according to selection criteria which were divided into 2 groups: patients with micronized progesterone expels and patients exposed to caproate. 17 dihydroxyprogesterone; applying the relative risk, and the chi square statistical test. Results: The frecuency of abortion in patients exposed to micronized natural progesterone was 22%. The frequency of abortion in patients exposed to caproate 17 dihydroxyprogesterone was 42%. The effectiveness of micronized natural progesterone is superior to that of caproate 17 dihydroxyprogesterone in the managemetn of threatened abortion with a RR=1.34 (p<0.005). Conclusion: Micronized natural progesterone therapy is superior to caproate 17 dihydroxyprogesterone in the management of threatened abortion.Tesi