Martin Štícha

Induction of cancer cell apoptosis by α-tocopheryl succinate: molecular pathways and structural requirements

The vitamin E analog α‐tocopheryl succinate (α‐TOS) can induce apoptosis. We show that the proapoptotic activity of α‐TOS in hematopoietic and cancer cell lines involves inhibition of protein kinase C (PKC), since phorbol myristyl acetate prevented α‐TOS‐triggered apoptosis. More selective effectors indicated that α‐TOS reduced PKCα isotype activity by increasing protein phosphatase 2A (PP2A) activity. The role of PKCα inhibition in α‐TOS‐induced apoptosis was confirmed using antisense oligonucleotides or PKCα overexpression. Gain‐ or loss‐of‐function bcl‐2 mutants implied modulation of bcl‐2 activity by PKC/ PP2A as a mitochondrial target of α‐TOS‐induced proapoptotic signals. Structural analogs revealed that α‐tocopheryl and succinyl moieties are both required for maximizing these effects. In mice with colon cancer xenografts, α‐TOS suppressed tumor growth by 80%. This epitomizes cancer cell killing by a pharmacologically relevant compound without known side effects.—Neuzil, J., Weber, T., Schröder, A., Lu, M., Ostermann, G., Gellert, N., Mayne, G. C., Olejnicka, B., Nègre‐Salvayre, A., Stícha, M., Coffey, R. J., Weber, C. Induction of cancer cell apoptosis by α‐tocopheryl succinate: molecular pathways and structural requirements. FASEB J. 15, 403‐415 (2001)

A fast derivatization procedure for gas chromatographic analysis of perfluorinated organic acids.

A rapid and simple derivatization procedure has been developed for gas chromatographic determination of perfluorinated organic acids (PFCAs, C(6)-C(12)), using isobutyl chloroformate (IBCF) to convert the acids into the more volatile isobutyl esters, under catalysis by pyridine. The procedure was optimized in an acetonitrile medium and applied to GC techniques with electron-capture detection (GC-ECD) and mass spectrometry with electron-impact ionization (GC-EI-MS); for the sake of comparison, HPLC with electrospray-ionization MS (HPLC-ESI(-)-MS) was also tested. The LOD and LOQ values obtained for these three techniques were compared, and the lowest LODs were obtained with GC-ECD (0.06-1.80 microg mL(-1)). The procedure was further optimized in an aqueous medium, obtaining the best results in a phosphate buffer (pH 2.5, 50 mmol L(-1)), in which the LOD and LOQ values were measured for GC-ECD a GC-EI-MS. The lowest LODs were found for GC-EI-MS (0.030-0.314 microg mL(-1)). The practical applicability was tested on Vltava river water samples.

Enatiomeric determination of tramadol and O-desmethyltramadol in human urine by gas chromatography-mass spectrometry

A GC-MS assay for stereoselective determination of tramadol and its pharmacologically active phase I metabolite O-desmethyltramadol in human urine was developed. Nefopam was used as internal standard. The method involves a simple solid phase extraction with chiral analysis by gas chromatography-electron ionization mass spectrometry using m/z 263; 58, 249; 58, and 179; 58 for the determination of concentration of tramadol, O-desmethyltramadol and internal standard, respectively. Chromatography was performed on a Rt-betaDEXcst column containing alkylated beta-cyclodextrins as a chiral selector. The calibration curves were linear in the concentration range 0.1-20 microg/mL (R(2) > or =0.998). Intra-day accuracies ranged between 97.2-104.9%, 96.1-103.2%, and 97.3-102.8% at the lower, intermediate, and high concentration for all analytes, respectively. Inter-day accuracies ranged between 95.2-105.7%, 99.1-105.2%, and 96.5-101.2% at the lower, intermediate, and high concentration for all analytes, respectively. This method was successfully used to determine the concentration of enantiomers of T and ODT in a pharmacogenetic study.

Ubiquinone-binding site mutagenesis reveals the role of mitochondrial complex II in cell death initiation

Respiratory complex II (CII, succinate dehydrogenase, SDH) inhibition can induce cell death, but the mechanistic details need clarification. To elucidate the role of reactive oxygen species (ROS) formation upon the ubiquinone-binding (Qp) site blockade, we substituted CII subunit C (SDHC) residues lining the Qp site by site-directed mutagenesis. Cell lines carrying these mutations were characterized on the bases of CII activity and exposed to Qp site inhibitors MitoVES, thenoyltrifluoroacetone (TTFA) and Atpenin A5. We found that I56F and S68A SDHC variants, which support succinate-mediated respiration and maintain low intracellular succinate, were less efficiently inhibited by MitoVES than the wild-type (WT) variant. Importantly, associated ROS generation and cell death induction was also impaired, and cell death in the WT cells was malonate and catalase sensitive. In contrast, the S68A variant was much more susceptible to TTFA inhibition than the I56F variant or the WT CII, which was again reflected by enhanced ROS formation and increased malonate- and catalase-sensitive cell death induction. The R72C variant that accumulates intracellular succinate due to compromised CII activity was resistant to MitoVES and TTFA treatment and did not increase ROS, even though TTFA efficiently generated ROS at low succinate in mitochondria isolated from R72C cells. Similarly, the high-affinity Qp site inhibitor Atpenin A5 rapidly increased intracellular succinate in WT cells but did not induce ROS or cell death, unlike MitoVES and TTFA that upregulated succinate only moderately. These results demonstrate that cell death initiation upon CII inhibition depends on ROS and that the extent of cell death correlates with the potency of inhibition at the Qp site unless intracellular succinate is high. In addition, this validates the Qp site of CII as a target for cell death induction with relevance to cancer therapy.

Polymerization of p-nitrophenylacetylene with metathesis catalysts. Photoelectrical properties of phenylacetylene/p-nitrophenylacetylene copolymer

Preparation of substituted acetylene polymers carrying nitro group is reported for the first time. Homopolymers of p-nitrophenylacetylene (NPA) and its copolymer with phenylacetylene (PA) have been prepared by polymerizations induced by WOCl 4 /3Me 4 Sn as a catalyst in benzene and benzene/dioxane as solvents. WOCl 4 alone does not polymerize, oligomerize and/or cyclotrimerize NPA. The two-component catalysts WOCl 4 /2Ph4Sn and MoOCl 4 /3Me 4 Sn produce low amounts of oligomers only. The homopolymer of NPA is insoluble, whereas the copolymer containing one NPA unit per 4.38 units of PA is soluble in aromatic solvents and THF. The IR and NMR spectra of the homopolymer and the IR spectrum of the copolymer are reported. The photoconductive properties of the copolymer are also reported and compared with those of poly(PA). Introduction of NO 2 groups onto polymer chains was found to increase the quantum efficiency of charge carrier photogeneration but to deteriorate the charge carrier transport properties of the polymer.

The biological effects of bilirubin photoisomers

Although phototherapy was introduced as early as 1950’s, the potential biological effects of bilirubin photoisomers (PI) generated during phototherapy remain unclear. The aim of our study was to isolate bilirubin PI in their pure forms and to assess their biological effects in vitro. The three major bilirubin PI (ZE- and EZ-bilirubin and Z-lumirubin) were prepared by photo-irradiation of unconjugated bilirubin. The individual photoproducts were chromatographically separated (TLC, HPLC), and their identities verified by mass spectrometry. The role of Z-lumirubin (the principle bilirubin PI) on the dissociation of bilirubin from albumin was tested by several methods: peroxidase, fluorescence quenching, and circular dichroism. The biological effects of major bilirubin PI (cell viability, expression of selected genes, cell cycle progression) were tested on the SH-SY5Y human neuroblastoma cell line. Lumirubin was found to have a binding site on human serum albumin, in the subdomain IB (or at a close distance to it); and thus, different from that of bilirubin. Its binding constant to albumin was much lower when compared with bilirubin, and lumirubin did not affect the level of unbound bilirubin (Bf). Compared to unconjugated bilirubin, bilirubin PI did not have any effect on either SH-SY5Y cell viability, the expression of genes involved in bilirubin metabolism or cell cycle progression, nor in modulation of the cell cycle phase. The principle bilirubin PI do not interfere with bilirubin albumin binding, and do not exert any toxic effect on human neuroblastoma cells.

HPLC/MS Analysis of Historical Pharmaceutical Preparations of Heroin and Cocaine

Pharmaceutical preparations of heroin and cocaine more than seventy years old were analyzed using RP-HPLC. The composition of mobile phase was optimized. The components were identified by MS2 or MS3, and the APPI fragmentation mechanisms of compounds found were proposed. The sample of heroin hydrochloride injection solution consists of 96.1% morphine and 3.9% of codeine. The sample of cocaine hydrochloride injection solution consists of 26.9% cocaine, 31.5% benzoylecgonine, 17.4% ecgonine, and 24.2% ecgonine methyl ester.

STRUCTURE-PROPERTY RELATIONSHIPS OF THIOACRIDINES; THEIR ELECTROCHEMICAL OXIDATION AS A MODEL OF METABOLIC DEGRADATION

ABSTRACT The relationships of structure vs. electrochemical and vs. chromatographical properties of thirteen 9-(alkylthio)acridines were studied and quantified by correlation equations between E 1/2 (t r , resp.), and substituent constants. The study of electrochemical oxidation of these compounds as the model of their possible metabolic degradation was performed. The oxidation products were separated and analysed by mass spectrometry. The probable scheme of electrochemical oxidation of studied derivatives was proposed.

Characterization of Rhenium(V) Complexes with Phenols Using Mass Spectrometry with Selected Soft Ionization Techniques

The synthesis, characterization, and mass spectra of oxorhenium(V) complexes with 1,2-dihydroxybenzene, 1,2,3-trihydroxybenzene, and 2,3-dihydroxynaphtalene are reported. Electrospray ionization, atmospheric pressure photoionization, and laser desorption/ionization mass spectra of the complexes showed abundant negatively charged molecular anions and low fragmentation. Calculated similarity indexes showed significant conformity between the computed and experimental isotopic patterns of selected ions and confirmed correct assignment of elemental composition to m/z values. Electrospray tandem mass spectrometry provided essential information about fragments from molecular ions of studied complexes, making it possible to distinguish among fragment ions and the ions arising from compounds present in the reaction mixture. Based on the results, mass spectrometry utilizing soft common ionization techniques is useful for monitoring complex formation reaction kinetics and the stabilities of the complexes. Representative spectra were recorded for micromolar concentrations of the analytes.

Development of a fast LC–MS/MS method for quantification of rilmenidine in human serum: elucidation of fragmentation pathways by HRMS

Rilmenidine is an alpha 2 adrenoreceptor agonist used in the treatment of mild and moderate hypertension. In this study, a fast and accurate liquid chromatographic method with tandem mass spectrometric detection has been validated in order to assure quantification of rilmenidine in human serum. The fragmentation pathway of protonated rilmenidine was studied using high-resolution mass spectrometry (HRMS). This study compared selectivity, linearity, accuracy, precision, extraction efficiency, matrix effect and sensitivity using common liquid-liquid extraction (LLE) and solid-phase extraction (SPE) procedures. The limit of quantitation for both extraction techniques was 0.1 ng/ml. Several differences between the LLE and SPE have been observed in terms of linearity, accuracy, precision and matrix effect. Additionally, the advantages of SPE included less manual work load and increased recovery of rilmenidine in human serum to approximately 80% (LLE, 57%). The developed method involving SPE was found to be accurate (relative error (RE) < 5%), reproducible (relative standard deviation, RSD < 7%), robust and suitable for quantitative analysis of rilmenidine in serum samples obtained from patients under antihypertensive treatment.