Controlled Release of Vanadium from a Composite Scaffold Stimulates Mesenchymal Stem Cell Osteochondrogenesis.

Large bone defects often require the use of autograft, allograft, or synthetic bone graft augmentation; however, these treatments can result in delayed osseous integration. A tissue engineering strategy would be the use of a scaffold that could promote the normal fracture healing process of endochondral ossification, where an intermediate cartilage phase is later transformed to bone. This study investigated vanadyl acetylacetonate (VAC), an insulin mimetic, combined with a fibrous composite scaffold, consisting of polycaprolactone with nanoparticles of hydroxyapatite and beta-tricalcium phosphate, as a potential bone tissue engineering scaffold. The differentiation of human mesenchymal stem cells (MSCs) was evaluated on 0.05 and 0.025 wt% VAC containing composite scaffolds (VAC composites) in vitro using three different induction media: osteogenic (OS), chondrogenic (CCM), and chondrogenic/osteogenic (C/O) media, which mimics endochondral ossification. The controlled release of VAC was achieved over 28 days for the VAC composites, where approximately 30% of the VAC was released over this period. MSCs cultured on the VAC composites in C/O media had increased alkaline phosphatase activity, osteocalcin production, and collagen synthesis over the composite scaffold without VAC. In addition, gene expressions for chondrogenesis (Sox9) and hypertrophic markers (VEGF, MMP-13, and collagen X) were the highest on VAC composites. Almost a 1000-fold increase in VEGF gene expression and VEGF formation, as indicated by immunostaining, was achieved for cells cultured on VAC composites in C/O media, suggesting VAC will promote angiogenesis in vivo. These results demonstrate the potential of VAC composite scaffolds in supporting endochondral ossification as a bone tissue engineering strategy.

High-performance liquid chromatographic analysis of dehydroepiandrosterone.

Qualitative and quantitative analysis of dehydroepiandrosterone and its conjugates in biological matrices and establishment of their relationships with physiological functions is a very active field. This review article discusses methods of separation and quantification of dehydroepiandrosterone and its conjugates using high-performance liquid chromatographic techniques.

Ergosteroids IV: synthesis and biological activity of steroid glucuronosides, ethers, and alkylcarbonates.

The 7-oxo derivative of dehydroepiandrosterone is more active than the parent steroid and is devoid of adverse side effects in rats, monkeys and humans. In anticipation of possible therapeutic use we have sought more active, longer lasting forms of 7-oxo- and 7beta-hydroxydehydroepiandrosterones. The 7-oxo- and 7-hydroxy steroids have been converted to glucuronides, ethers and carbonate esters. The syntheses of these compounds are described and their ability to induce the formation of liver thermogenic enzymes when fed to rats is reported. Some of the new derivatives were found to be somewhat more effective than the equimolar amounts of 7-oxo-DHEA with which they were compared in each experiment.

Steroidal allylic fluorination using diethylaminosulfur trifluoride: a convenient method for the synthesis of 3 beta-acetoxy-7 alpha- and 7 beta-fluoroandrost-5-en-17-one.

This paper discusses our findings regarding fluorination of the diastereomeric 3 beta-acetoxy-7-hydroxyandrost-5-en-17-ones (3 and 4) at the allylic 7-hydroxyl group using diethylaminosulfur trifluoride under various experimental conditions. The reaction led to the formation of allylic 7 alpha- and 7 beta-fluoro derivatives, 6 and 7, contaminated with small amounts of 3 beta-acetoxy-5 alpha-fluoroandrost-6-en-17-one (8), the rearrangement product, and 3 beta-acetoxyandrosta-4,6-dien-17-one (9), the elimination product. However, synthesis of 3 beta-acetoxy-7 alpha-fluoroandrost-5-en-17-one (6) and 3 beta-acetoxy-7 beta-fluoroandrost-5-en-17-one (7) has been achieved in high isomeric purity by careful manipulation of the experimental conditions. Also included herein is a convenient chemical synthesis of pure 3 beta-acetoxy-7 alpha-hydroxyandrost-5-en-17-one (4) and 3 beta-acetoxy-7 beta-hydroxyandrost-5-en-17-one (3), the starting materials for the present fluorination reaction. The structure of a degradation product, 3 beta-acetoxy-5 alpha-hydroxyandrost-6-en-17-one (5), has been established by X-ray diffraction analysis to ascertain unambiguously its absolute configuration.

Ergosteroids. II: Biologically active metabolites and synthetic derivatives of dehydroepiandrosterone.

An improved procedure for the synthesis of 3 beta-hydroxyandrost-5-ene-7,17-dione, a natural metabolite of dehydroepiandrosterone (DHEA) is described. The synthesis and magnetic resonance spectra of several other related steroids are presented. Feeding dehydroepiandrosterone to rats induces enhanced formation of several liver enzymes among which are mitochondrial sn-glycerol 3-phosphate dehydrogenase (GPDH) and cytosolic malic enzyme. The induction of these two enzymes, that complete a thermogenic system in rat liver, was used as an assay to search for derivatives of DHEA that might be more active than the parent steroid. Activity is retained in steroids that are reduced to the corresponding 17 beta-hydroxy derivative, or hydroxylated at 7 alpha or 7 beta, and is considerably enhanced when the 17-hydroxy or 17-carbonyl steroid is converted to the 7-oxo derivative. Several derivatives of DHEA did not induce the thermogenic enzymes whereas the corresponding 7-oxo compounds did. Both short and long chain acyl esters of DHEA and of 7-oxo-DHEA are active inducers of the liver enzymes when fed to rats. 7-Oxo-DHEA-3-sulfate is as active as 7-oxo-DHEA or its 3-acetyl ester, whereas DHEA-3-sulfate is much less active than DHEA. Among many steroids tested, those possessing a carbonyl group at position 3, a methyl group at 7, a hydroxyl group at positions 1, 2, 4, 11, or 19, or a saturated B ring, with or without a 4-5 double bond, were inactive.

Ergosteroids III. Syntheses and biological activity of seco-steroids related to dehydroepiandrosterone.

The unusual activity of some D-ring-seco estrogens led us to prepare several seco steroids related to dehydroepiandrosterone (DHEA) and to test for their ability to mimic thyroid hormone and 7-oxo-DHEA (1) as inducers of thermogenic enzymes in rats' livers. Only one, 3 beta-acetoxy-17a-oxa-androst-5-ene-7,17-dione (17), was capable of inducing both mitochondrial glycerophosphate dehydrogenase and malic enzyme. The closely related 3 beta-hydroxy-17a-oxa-androsta-5,15-diene-7,17-diones (both 14 alpha and 14 beta, 14 and 15) induce the formation of malic enzyme but not of glycerophosphate dehydrogenase. The 3 beta-propionyl ester of the above 14 alpha steroid was not active, presumably because it was not deacylated in vivo. The 16,17 dicarboxylic acid (9) produced by opening the D-ring also induced the formation of malic enzyme but not of glycerophosphate dehydrogenase. 3 beta-Acetoxyandrost-5-ene-7,16,17-trione, an intermediate in the synthesis of D-ring seco compounds enhanced the formation of both enzymes. Twelve other D-ring seco compounds were not active. Seco androstanes oxygenated at position 7 and with expanded A or B rings were not active.

Liquid chromatography-electrospray ionization mass spectrometric analysis of corticosterone in rat plasma using selected ion monitoring.

A simple and fast yet highly sensitive and specific method based on HPLC coupled to electrospray ionization mass spectrometry has been developed for the quantitation of corticosterone in rat plasma. After extraction of rat plasma (100 microl) with diethyl ether using 5-pregnen-3beta-ol-20-one-16alpha-carbonitrile (Sigma) as internal standard, HPLC was performed on a short C8 column (Zorbax-Eclipse, 50x4.6 mm I.D.) using a steep methanol-water gradient (methanol 54% to 90% in 6 min). Detection was performed on a single quadruple mass spectrometer in selected ion monitoring mode (m/z 369 for corticosterone and 364 for the internal standard). The detection limit of the assay was 9 fmol (3 pg) of corticosterone on column. In vitro data were subjected to curve fitting (cubic, r2=0.9999). Recovery of corticosterone after extraction ranged from 81 to 93%. The relative standard deviations for intra- and inter-assay precision ranged from 0.8 to 3.6% and 5.2 to 12.9%, respectively. Corticosterone did not undergo any appreciable degradation when stored in plasma at -20 degrees C for 2 months. The assay is routinely used in our laboratory to examine corticosterone levels as a marker of stress in rats and may also be used for the determination of 18-hydroxy-11-deoxycorticosterone.

Development and validation of a high-performance liquid chromatography assay for the quantitative determination of 7-oxo-dehydroepiandrosterone-3beta-sulfate in human plasma.

A new, simple, reproducible and reliable high-performance liquid chromatography method with ultraviolet absorbance detection at 240 nm was developed and validated for the determination of 7-oxo-dehydroepiandrosterone-3beta-sulfate in human plasma. The method was based upon solid-phase (C18) extraction of plasma after addition of 17beta-hydroxy-3beta-methoxyandrost-5-en-7-one as internal standard. Using 1 ml of plasma for extraction, the detection limit of the assay was 3 ng/ml. The standard curve was linear over the concentration range 10-1000 ng/ml. Stored at -20 degrees C for about 4 months at various concentrations in plasma, 7-oxo-dehydroepiandrosterone-3beta-sulfate did not reveal any appreciable degradation. Also included herein is a method for the simultaneous detection and determination of 7-oxo-dehydroepiandrosterone and 7-oxo-dehydroepiandrosterone-3beta-acetate in plasma.

Effect of pregnancy anaemia on cellular growth in the human placenta.

The effects of pregnancy anaemia on cell number and size in human placentae were investigated. Total organ weight, protein, RNA and DNA contents were determined in 54 fresh placentae from anaemic women (haemoglobin less than 110 g/l) and another 32 placentae from women without anaemia (haemoglobin greater than or equal to 110 g/l). The placental weight was significantly reduced in pregnancy anaemia. The decrease in total placental DNA in anaemic women suggested that the reduced placental weight was due to a decrease in cell number. However, these placentae also showed evidence of compensatory cellular hypertrophy as indicated by increase in both weight per cell and protein per cell.

Safety and pharmacokinetic study with escalating doses of 3-acetyl-7-oxo-dehydroepiandrosterone in healthy male volunteers.

OBJECTIVES: To evaluate the safety and pharmacokinetics of 3-acetyl-7-oxo-DHEA (3beta-acetoxyandrost-5-ene-7,17-dione) given orally. DESIGN: A randomized, double blind, placebo-controlled, escalating dose study. SETTING: The Chicago Center for Clinical Research. PARTICIPANTS: Twenty-two healthy men. STUDY METHOD: The participants received placebo (n = 6) or 3-acetyl-7-oxo-DHEA (n = 16) at 50 mg/d for 7 days followed by a 7-day washout; 100 mg/d for 7 days followed by a 7-day washout; and 200 mg/d for 28 days. OUTCOME MEASURES: Safety parameters, evaluated at each dose level, included measurement of total testosterone, free testosterone, dihydrotestosterone, estradiol, cortisol, thyroxin and insulin levels. Analyses for 7-oxo-DHEA-3beta-sulfate (DHEA-S), the only detectable metabolic product of the administered steroid, were conducted on plasma drawn from all subjects at 0.25, 0.5, 1, 2, 4, 6 and 12 hours after the final 100 mg dose of 3beta-acetyl-7-oxo-DHEA. RESULTS: There were no differences in the clinical laboratory values or in reported minor adverse experiences, between treatment and placebo groups. In general, blood hormone concentrations were unaffected by the treatment with 3beta-acetyl-7-oxo-DHEA and remained within the normal range. No changes in vital signs, blood chemistry or urinalysis occurred during treatment with 3beta-acetyl-7-oxo-DHEA compared to placebo. The administered steroid was not detected in the blood but was rapidly converted to 7-oxo-DHEA-S, the concentrations of which were proportional to dose. This steroid sulfate did not accumulate; plasma concentrations 12 hours after the 3beta-acetyl-7-oxo-DHEA dose at 7 and 28 days on the 200 mg/d dose were 15.8 and 16.3 microg/L respectively. The mean time to peak plasma level of 7-oxo-DHEA-S was 2.2 hours; the mean half life was 2.17 hours. The apparent clearance averaged 172 L/h, and the apparent mean volume of distribution was 540 L. CONCLUSION: These results indicate that 3beta-acetyl-7-oxo-DHEA is safe and well tolerated in normal healthy men at doses up to 200 mg/d for 4 weeks.

Suppression of Delta(5)-androstenediol-induced androgen receptor transactivation by selective steroids in human prostate cancer cells.

Our earlier report suggested that androst-5-ene-3beta,7beta-diol (Delta(5)-androstenediol or Adiol) is a natural hormone with androgenic activity and that two potent antiandrogens, hydroxyflutamide (Eulexin) and bicalutamide (Casodex), fail to block completely the Adiol-induced androgen receptor (AR) transactivation in prostate cancer cells. Here, we report the development of a reporter assay to screen several selective steroids with anti-Adiol activity. Among 22 derivatives/metabolites of dehydroepiandrosterone, we found 4 steroids [no. 4, 1,3,5(10)-estratriene-17alpha-ethynyl-3, 17beta-diol; no. 6, 17alpha-ethynyl-androstene-diol; no. 8, 3beta, 17beta-dihydroxy-androst-5-ene-16-one; and no. 10, 3beta-methylcarbonate-androst-5-ene-7,17-dione] that have no androgenic activity and could also block the Adiol-induced AR transactivation in prostate cancer PC-3 cells. Interestingly, these compounds, in combination with hydroxyflutamide, further suppressed the Adiol-induced AR transactivation. Reporter assays further showed that these four anti-Adiol steroids have relatively lower glucocorticoid, progesterone, and estrogenic activity. Together, these data suggest some selective steroids might have anti-Adiol activity, which may have potential clinical application in the battle against the androgen-dependent prostate cancer growth.