Helmut Gleispach

The use of different silylating agents for structure analyses of steroids

Abstract A number of different silylating agents and their mixtures are available with different activities and, if combined, mutual stimulation or even suppression of their behaviour may occur. We were therefore interested in determining the optimal reaction conditions and also the possibility of using them for structure analyses. Different steroids have been treated with silylating agents under various conditions. The silyl ethers were dissolved in carbon tetrachloride and analysed by gas—liquid chromatography—mass spectrometry. It could be proved that every hydroxyl group in a steroid could be transformed into its silyl ether depending on the silylating mixture used. Oxo groups in the α-position to a hydroxyl group can react with formation of the enediol trimethylsilyl ether, whereas free oxo groups react under strong conditions with formation of the enol trimethylsilyl derivative.

Extracellular Ca2+ sensing by the osteoblast-like cell line, MC3T3-E1.

The present study was undertaken to clarify the role of extracellular calcium on osteoblast activation. It was found that bradykinin and thrombin induced synthesis of prostaglandin E2 was strongly dependent on the concentration of extracellular calcium in the osteoblast-like cell line, MC3T3-E1. Moreover, this effect was not related to Ca2+ influx, since it was even potentiated by Ni2+ and Co2+, which was not due to intracellular activity of Ni2+, as judged by studies with 63Ni2+. Ba2+, Mg2+ and Sr2+ had no effect. Cd2+ caused dose-dependent synthesis of prostaglandin E2, which was shown to correlate with its cytotoxic properties. The results thus strongly suggest the presence of a divalent cation sensor in osteoblast-like MC3T3-E1 cells.

On the inhibition of prostanoid formation by SK&F 96365, a blocker of receptor‐operated calcium entry

1 The proposed blocker of receptor‐operated calcium channels, SK&F 96365 was shown to inhibit formation of prostaglandin E2 in two osteoblast‐like cell lines, MC3T3‐E1 and UMR‐106 in a dose‐dependent manner at an IC50 of 3–4 μm. Inhibition was observed with various stimuli (arachidonic acid, bradykinin and calcium ionophore A23187). 2 This effect was also observed in human platelets, where SK&F 96365 dose‐dependently blocked thromboxane biosynthesis and formation of 12‐hydroxy‐heptadecatrienoic acid after stimulation with arachidonic acid (IC50 = 4 μm). 3 The compound had no effect on 12‐hydroxy‐eicosatetraenoic acid production by human platelets. Additionally, linoleic acid oxidation by soybean 15‐lipoxidase was not impaired by SK&F 96365. The results thus provide evidence for cyclo‐oxygenase inhibition by SK&F 96365 at concentrations used to block receptor‐operated calcium influx.

Isotope dilution gas chromatography—mass spectrometry for the study of eicosanoid metabolism in human blood platelets

Stable isotope dilution gas chromatography-mass spectrometry provides one of the most important techniques for the quantitative measurement of eicosanoids. This technique was applied to the quantitation of hydroxyeicosatetraenoic acids, hydroxyheptadecatrienoic acid, thromboxane B2 and prostaglandin F2 alpha formed during platelet aggregation after stimulation of gel-filtered platelets with thrombin (0.25 U/ml) or collagen (2 micrograms/ml). Similar amounts of hydroxyheptadecatrienoic acid and thromboxane B2 were found after platelet activation. The ratio of formation of 12-hydroxyeicosatetraenoic acid to thromboxane B2 varied from donor to donor. Only small amounts of prostaglandin F2 alpha (up to 200 pg per 2.0.10(8) platelets) and basic values of 15-hydroxyeicosatetraenoic acid (up to 100 pg per 2.0.10(8) platelets) were measured using gas chromatography with negative ion chemical ionization mass spectrometry. In addition, different stable isotope dilutions were prepared and are discussed in detail.

Digoxin elimination by exchange transfusion.

The report covers four cases presenting simultaneous indications for digitalisation and exchange transfusions. Intravenous administration of digoxin was followed: 1. by monitoring of the behaviour of the plasma digoxin level; 2. by determination of the total amount of glycoside eliminated by the blood exchange. Particular attention was paid to the effect of the delay between injection and exchange transfusion on the amount of digoxin eliminated.All four cases showed moderate falls in plasma levels. The amounts of digoxin eliminated by exchange transfusion were in reverse relationship to the delay between administration of digoxin and the blood exchange. At no time did the eliminated fraction exceed 5% of the total amount present in the body.ZusammenfassungEs wird von vier Fällen berichtet, bei denen sich zugleich die Indikation zur Digitalisierung und zur Austauschtransfusion ergab. Nach vorhergehender, intravenöser Digoxingabe wurde einerseits das Verhalten der Digoxinkonzentration im Plasma gemessen, andererseits die gesamte durch den Blutaustausch eliminierte Glykosidmenge bestimmt. Besondere Beachtung galt einer Abhängigkeit des eliminierbaren Prozentsatzes von dem zeitlichen Abstand der Austauschtransfusion zur vorangehenden Injektion.In allen vier Fällen wurde ein mäßiges Absinken der Plasmakonzentration beobachtet. Die durch Austauschtransfusion eliminierte Digoxinmenge stand in einem verkehrt proportionalen Verhältnis zum Intervall zwischen Digoxingabe und Blutaustausch. In keinem Fall überstieg diese eliminierte Fraktion 5% der insgesamt im Körper enthaltenen Menge.

Preparation of Deuterated Diclofenac for Use as an Internal Standard in Quantitative Measurement by Gas Chromatography Negative‐ion Chemical Ionization Mass Spectrometry

A procedure for the preparation of deuterated diclofenac is described. The resulting d5-diclofenac was used as an internal standard for quantitative measurement of the drug. The benefits of deuteration versus oxygen-18 labeling are demonstrated. Enhanced stability of the isotope label resulted in greater flexibility in sample handling during the work-up steps of plasma samples.

Inhibition of prostanoid formation in intact cells by 2,5‐di‐(tert‐butyl)‐1,4‐benzohydroquinone, a blocker of Ca2+‐ATPases

1 The blocker of endoplasmic reticulum Ca2+‐ATPase, 2,5‐di‐(tert‐butyl)‐1,4‐benzohydroquinone (BHQ) was shown to inhibit formation of prostaglandin E2 and prostacyclin in the osteoblast‐like cell lines, MC3T3‐E1 and ROS 17/2.8, respectively, in a dose‐dependent manner with an IC50 of 0.5‐1 μm. Inhibition was observed with various stimuli (arachidonic acid, bradykinin, melittin and calcium ionophore, A23187). 2 This effect was also observed in human platelets, where BHQ dose‐dependently blocked thromboxane biosynthesis and formation of 12‐hydroxy‐heptadecatrienoic acid after stimulation with arachidonic acid, but not formation of 12‐hydroxy‐eicosatetraenoic acid. 3 Inhibition of prostaglandin E2 formation in MC3T3‐E1 cells was not observed with thapsigargin after stimulation with arachidonic acid, A23187 or melittin, whereas bradykinin‐induced prostaglandin E2 biosynthesis was blocked. 4 Taken together, the results suggest a direct inhibitory action of BHQ on the cyclo‐oxygenase in these three cell systems.

Applications of gas chromatography-mass spectrometry in clinical chemistry

Two applications of gas chromatography-mass spectrometry in clinical chemistry, are described, namely the identification and determination of compounds present in biological fluids. For the first application, the biological substances are derivatized to stable forms for gas chromatography and to give appropriate spectra in the mass spectrometer operated in the scanning mode. This method is used for the diagnosis of organic acidurias, different enzyme deficiencies causing congenital adrenal hyperplasia and other disturbances of steroid metabolism, for differentiation between adrenal carcinoma or adenoma, etc. The second application is the use of the mass spectrometer as a very sensitive and selective gas chromatographic detector. This, in combination with stable isotope dilution methods, is the most accurate analytical method in clinical chemistry. The substances are derivatized to forms suitable for gas chromatography and to give a few specific and intense fragment ions in the mass spectrometer operated in selected-ion monitoring mode. Substances that can be detected in this way include steroids, vitamins, prostaglandins, carbohydrates, drugs and pesticides.

Heparin Inhibition of the Cytostatic Effect of Adriamycin® on Cultured Lymphocytes

The cytostatic effects of Adriamycin® on PHA-stimulated lymphocyte incubates are inhibited by the simultaneous administration of heparin. Simultaneous administration of heparin, therefore, might also

Formation of 6,15-diketoprostaglandin F1α from prostaglandin G2 by bovine aortic endothelial cells

Besides 6-ketoprostaglandin F1 alpha, bovine aortic endothelial cells also produced considerable amounts of 6,15-diketoprostaglandin F1 alpha from arachidonic acid, either exogenously added or released from cellular phospholipids. Incubations of particulate fractions of endothelial cells with the cyclic endoperoxides prostaglandin G2 and prostaglandin H2 showed that 6,15-diketoprostaglandin F1 alpha is formed by the action of prostaglandin I2 synthetase on prostaglandin G2. The labile metabolite 15-hydroperoxyprostaglandin I2 is then converted nonenzymatically to the 15-keto derivative. In the presence of reduced glutathione, quantitative analysis of both metabolites by gas chromatography-mass spectrometry showed a significant decrease of 6,15-diketoprostaglandin F1 alpha formation, whereas prostaglandin I2 synthesis was markedly increased. This shift seems to be due to a stimulation of peroxidase by GSH, a well known cofactor of this enzyme. Thus, it seems that a decreased endothelial prostaglandin I2 formation may occur when cellular glutathione levels are reduced as a consequence of oxidant injury and lipid peroxidation. Additionally, ferrous ions seems to be involved in the regulation of endothelial prostaglandin I2 synthesis, since Desferal, a specific ferrous ion chelator that might have antimetastatic properties, produced a pronounced shift from 6,15-diketoprostaglandin F1 alpha to the 6-keto derivative, i.e., prostaglandin I2.