Hans Jörg Leis

Phenotypic Heterogeneity of Osteoblast-like MC3T3-E1 Cells: Changes of Bradykinin-Induced Prostaglandin E2 Production During Osteoblast Maturation

We have examined clonal murine calvarial MC3T3‐E1 cells obtained from different sources to compare their osteoblastic features (alkaline phosphatase [ALP], cyclic adenosine monophosphate [cAMP] response to parathyroid hormone, prostaglandin E2 (PGE2) and PGE1, bradykinin‐induced production of PGE2). It was found that the sublines investigated showed large variation of the above‐mentioned parameters, which may be attributed to distinct differentiated stages of osteoblast development. Increase of ALP activity was paralleled by an increase in cAMP accumulation in response to the above‐mentioned agents. The most striking difference was observed with bradykinin‐induced production of PGE2. Early stage cells (low ALP) produced high levels of PGE2, whereas cells with high ALP activity showed no bradykinin stimulation at all. This was consistent with the results of specific binding of3H‐bradykinin to its receptor and also correlated well with the bradykinin‐induced signal transduction sequence (inositol triphosphate liberation and elevation of intracellular calcium levels). This was confirmed by Northern blot analysis of bradykinin receptor mRNA expression. These results indicate that the widely used osteoblast‐like cell line MC3T3‐E1 is synonymous for multiple sublines, representing different stages of osteoblast development. These sublines were most likely emerging from the early stage cell line due to the applied culture conditions. Moreover, distinct biochemical features are displayed in correlation to the differentiation stage, thus providing a useful model to study the molecular mechanism of osteoblast maturation.

Negative ion chemical ionization for the determination of methylphenidate in human plasma by stable isotope dilution gas chromatography/mass spectrometry

A sensitive and specific method for the determination of methylphenidate in human plasma is presented. Methylphenidate was extracted from plasma by solvent extraction with hexane at pH 9.3 and derivatized to its heptafluorobutyrate derivative. The derivative was measured by gas chromatography/negative ion chemical ionization mass spectrometry without any further purification. Using this detection mode, a diagnostically useful fragment ion at m/z 369 was obtained at high relative abundance. (18)O(2)-labelled methylphenidate was used as an internal standard and its rapid and facile preparation from the unlabeled compound is described. Calibration graphs were linear within the range 0.14-18.25 ng ml(-1). The inter-assay precision was 8.7% (0.14 ng ml(-1)) and 3.1% (4.56 ng ml(-1)) and the intra-assay variability was 1.3% (0.14 ng ml(-1)) and 0.4% (4.56 ng ml(-1)). Accuracy determinations showed deviations of +0.7% (0.14 ng ml(-1)) and -2.5% (4.56 ng ml(-1)). The method is rugged, rapid and robust and has been applied to the batch analysis of methylphenidate during pharmacokinetic profiling of the drug.

Quantitative analysis of morphine in human plasma by gas chromatography-negative ion chemical ionization mass spectrometry

A sensitive and specific method for the quantitative determination of morphine in human plasma is presented. Morphine was extracted from plasma by solvent extraction with ethyl acetate and derivatized to its heptafluorobutyrate derivative. The derivatives were measured by gas chromatography-negative ion chemical ionization mass spectrometry without any further purification. Using this detection mode, a diagnostic useful fragment ion at m/z 637 is obtained at high relative abundance. Deuterated morphine was used as an internal standard. Calibration graphs were linear within a range of 0.78 ng/ml and 50 ng/ml. Inter-assay precision was 2.3% (2.85 ng/ml) and 1.4% (14.25 ng/ml), intra-assay variability was found to be 1.5% (3.71 ng/ml) and 0.5% (14.54 ng/ml). Accuracy showed deviations of -9.3% (2.85 ng/ml) and -4.2% (14.25 ng/ml). The method is rugged and robust and has been applied to the batch analysis of morphine during pharmacokinetic profiling of the drug.

Determination of captopril in human blood by gas chromatography—negative-ion chemical ionization mass spectrometry with [18O4]captopril as internal standard

A method for the quantitative measurement of captopril in human blood is described. Blood was immediately treated with N-ethylmaleimide to prevent oxidative degradation. The carboxyl moiety was derivatized to the pentafluorobenzyl ester, which shows excellent properties for negative-ion chemical ionization mass spectrometry. A stable isotope-labelled standard was prepared from the intact target molecule in quantitative yield by exchanging the oxygen atoms of the free carboxylic acid and the imide moiety against 18O. The detection limit under negative-ion chemical ionization conditions is ca. 100 times lower than under electron-impact or positive-ion chemical ionization conditions, therefore only very small amounts of the original sample have to be analysed. The method was applied to be quantitative determination of unchanged captopril in human plasma after oral administration of a 25-mg dose.

Synthesis of new 2-, 3-, and 4-substituted azidoquinolines: inhibitors of human blood platelet aggregation in vitro

Abstract A series of 2-, 3- and 4-substituted azidoquinoline derivatives were synthesized and tested for their ability to inhibit human platelet aggregation in vitro triggered by adenosine diphosphate (15 μM), collagen (5 μg/ml), platelet activating factor (10 μM), or the stable prostaglandin H2 mimetic, U46619 (4 μM). The most active compounds (IC50 2.5–68.3 μM) were the germinal 3,3-diazides (4f and 4g) and the 4-azido-3-nitroquinolines (6f and 6g).

Qualitative and quantitative measurement of hydroxy fatty acids, thromboxanes and prostaglandins using stable isotope dilutions and detection by gas chromatography—mass spectrometry

Methods for measurement of the metabolites of arachidonic acid (AA), namely prostaglandins (PGs), thromboxanes (TXs) and hydroxy fatty acids, using stable isotope dilution gas chromatography--mass spectrometry are described. With a few exceptions, labelled species of the various AA metabolites are not commercially available and were therefore synthesized in our laboratory. [2H8]AA, produced by deuteration of eicosatetraynoic acid, was used for comparing the metabolism of exogenously added and endogenously present AA in fibroblast cultures. After derivatization and catalytic hydrogenation, structure elucidation and quantification of the different hydroxy fatty acids was carried out by determination of the fragment ions resulting from alpha-cleavage at the site of the hydroxy function. During catalytic hydrogenation a significant hydrogen--deuterium exchange was observed. To eliminate this problem, 18O-labelled standards were prepared by exchanging the oxygen of the carboxylic acid group. The preparation and the use of hydroxy fatty acids, PGs and TXs labelled with 18O is described.

Method for the Determination of Vitamin K1(20) in Human Plasma by Stable Isotope Dilution/Gas Chromatography/Mass Spectrometry

A method for the quantitative determination of vitamin K1(20) (VK), an essential cofactor in the carboxylation of clotting factors, is presented. The assay is based on gas chromatography/electron impact mass/spectrometry. The preparation of deuterium-labelled vitamin K1(20) for use as an internal standard is described. The method involves extraction of VK from human plasma and its derivatization to the heptafluorobutyryl ester after reduction of one carbonyl group with zinc. The detection limit was found to be 1.0 pg and the limit of quantitation 2.0 pg ml-1 plasma. This permits the measurement of vitamin K1(20) even in small quantities of plasma, which is highly desirable in investigations dealing with clotting abnormalities in neonates and infants.

Improved sample preparation for the quantitative analysis of paroxetine in human plasma by stable isotope dilution negative ion chemical ionisation gas chromatography–mass spectrometry

An improved sample work-up and derivatisation procedure for the quantitative determination of paroxetine in human plasma by gas chromatography-negative ion chemical ionisation mass spectrometry is presented. Solvent extraction from plasma samples at alkaline pH was combined with derivatisation to the pentafluorobenzyl carbamate derivative in one step and subsequently analysed without any further purification. Thus, lengthy and time-consuming solvent evaporation steps are avoided to assure high-throughput analysis. Complete validation data are presented. The method is rugged, rapid and robust and has been applied to the batch analysis of paroxetine during pharmacokinetic profiling of the drug.

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.

Inhibition of cyclooxygenases 1 and 2 by the phospholipase-blocker, arachidonyl trifluoromethyl ketone

Arachidonyl trifluoromethyl ketone (ATK) is widely used as an inhibitor of cytosolic group IV phospholipase A2 (cPLA2) and calcium‐independent group VI phospholipase A2 (iPLA2). ATK thus reduces arachidonic acid (AA) substrate for cyclooxygenase (COX; also known as prostaglandin H synthase) and attenuates prostaglandin (PG) synthesis. It has been shown previously, that ATK blocks thromboxane B2 production induced by exogenous AA in human platelets. It remains, however, unknown whether ATK also directly modulates the activity of cyclooxygenase (COX).