Éva Szökő

Chiral separation of deprenyl and its major metabolites using cyclodextrin-modified capillary zone electrophoresis

A capillary electrophoretic method for the enantiomer resolution of deprenyl and its main alkaline metabolites amphetamine, methamphetamine and propargylamphetamine is described. An acidic separation buffer with a suitable chiral complexing agent, heptakis-(2,6-di-O-methyl)-β-cyclodextrin, was used and the optimum separation conditions were determined by changing the concentration of the chiral selector, the applied electric field and the concentration of methanol.

Determination of binding constants and the influence of methanol on the separation of drug enantiomers in cyclodextrin modified capillary electrophoresis

Abstract Binding constants of the optical isomers of phenylalkylamine derivatives and (2,6-di-O-methyl)-β-cyclodextrin were determined using electrophoretic mobility data gained from separations performed by capillary electrophoresis, and assuming a simple equilibrium model. Dependence of the optimum separation conditions and parameters on the binding constants were investigated and the influence of methanol was estimated.

Chiral analysis of amino acid neurotransmitters and neuromodulators in mouse brain by CE-LIF.

Chiral CE method has been developed for quantitative determination of d‐amino acid modulators of NMDA glutamate receptor; d‐serine and d‐aspartate along with l‐glutamate and l‐aspartate in biological samples. These ligands are suggested to be involved in regulation of NMDA receptor related brain functions, such as neurogenesis, neuronal plasticity, and memory formation. For sensitive determination of the amino acids LIF detection was chosen, and a fluorogenic reagent, 7‐fluoro‐4‐nitro‐2,1,3‐benzoxadiazole was used for derivatization. An amino‐modified β‐CD, 6‐monodeoxy‐6‐mono(3‐hydroxy)propylamino‐β‐CD (HPA‐β‐CD) was applied as chiral selector. Determinations were accomplished in a polyacrylamide coated capillary and reverse polarity was used for the analysis of the negatively charged analytes. The method was optimized and validated; 6 mM HPA‐β‐CD in 50 mM HEPES buffer, pH 7 was appropriate to achieve baseline separation of the analytes. The limit of quantification with acceptable accuracy is 0.05 μM for both d‐amino acids. The method was used for the determination of d‐aspartate and d‐serine content in various brain regions of adult mice.

R-Deprenyl: Pharmacological Spectrum of its Activity

Deprenyl has been discovered by Knoll and co-workers. The R-enantiomer of deprenyl (selegiline) is a selective and irreversible inhibitor of the B-isoform of monoamine oxidase (MAO-B) enzyme. Due to its dopamine potentiating and possible neuroprotective properties it has an established role in the treatment of parkinsonian patients. By inhibiting MAO-B enzyme, R-deprenyl decreases the formation of hydrogen peroxide, alleviating the oxidative stress also reduced by increased expression of antioxidant enzymes (superoxide dismutases and catalase) reported during chronic treatment. It was shown to prevent the detrimental effects of neurotoxins like MPTP and DSP-4. R-Deprenyl elicits neuroprotective and neuronal rescue activities in concentrations too low to inhibit MAO-B. It is extensively metabolized and some of the metabolites possess pharmacological activities, thus their contribution to neuroprotective properties was also suggested. The recently identified deprenyl-N-oxide is extensively studied in our laboratory. Effects other than neuroprotection, like influencing cell adhesion and proliferation cannot be neglected.

Deprenyl: from chemical synthesis to neuroprotection

During the last decades (-)-deprenyl has become the golden standard of MAO-B inhibitors. It possesses dopamine potentiating and antioxidant properties; however, its effects cannot be explained solely by the enzyme inhibitory action. (-)-Deprenyl prevents the toxicity of certain selective neurotoxins and recently it was demonstrated to increase cell-cell adhesion as well. The complexity of its pharmacological effects reflects the action of both the parent compound and the active metabolites. (-)-Deprenyl and related propargylamines (DRPs) show neuroprotective features in a variety of in vitro and in vivo models that is dependent on the propargyl moiety. The main presumptive targets to date include glyceraldehyde-3-phosphate dehydrogenase, poly(ADP-ribose) polymerase, some kinase cascades, as well as pro- and antiapoptotic proteins, beside the inhibition of MAO-B. The antiapoptotic activity of DRPs converges upon the maintenance of mitochondrial integrity, due to the initiation of a complex transcriptional program, the details of which are yet to be elucidated.

Comparison of quantitative performance of three fluorescence labels in CE/LIF analysis of aspartate and glutamate in brain microdialysate.

Three different fluorescent tags have been compared for the quantitative analysis of aspartate and glutamate in brain microdialysate samples. Separation conditions have been optimized to achieve short analysis time using reversed polarity separation in coated capillary. Method validation has revealed similar quantification limit of 0.1 μM of analytes using either of the labels, although LOD values were different: 7.8–9.8 nM for 4‐fluoro‐7‐nitro‐2,1,3‐benzoxadiazole, 3.5 nM for fluorescein‐5‐isothiocyanate and 1.3–1.5 nM for carboxyfluorescein succinimidyl ester derivatives. The almost two orders of magnitude difference between LOD and LOQ values is likely due to the unreliable derivatization reaction at low sample concentration. Based on the superior stability, FITC derivatization was used for the analysis of biological samples. The applicability of the method has been demonstrated by analyzing basal and potassium evoked amino acid concentrations in individual brain microdialysate samples.

Capillary gel electrophoretic separation of DNA restriction fragments in a discontinuous buffer system

Abstract Separation of single and double stranded DNA molecules by capillary gel electrophoresis has a rapidly growing interest. Similar to the polycrylamide slab gel based separation methods, in capillary gel electrophoresis, the paek/bans spacing usually decreases with the increasing size/length of the DNA molecule. Additionally, employing the regularly used Tris-borate buffer system, fronting peaks are often obtained. By the application of an electrolyte step gradient during capillary gel electrophoretic separation of dsDNA molecules, the apparent peak shape can be improved and the required analysis time decreased.

Chiral separations for d-amino acid analysis in biological samples.

It is widely accepted that some of the free d-amino acids play important biological role. d-Aspartate and d-serine formed in the central nervous system of higher vertebrates have neurotransmitter/neuromodulator function. Together with d-alanine they are distributed in various tissues and biological fluids. Studying their physiological and pathological significance requires their sensitive and accurate determination in biological samples. The various separation and detection methods used for their analysis are overviewed in the present paper. Our focus is mainly the quantitative performance and the analysis of real biospecimens.

Study on SSAO enzyme activity and anti-inflammatory effect of SSAO inhibitors in animal model of inflammation

SSAO/VAP-1 participates in the accumulation of leukocytes at the site of inflammation. A new SSAO inhibitor, SzV-1287 was demonstrated to inhibit both acute and chronic inflammation in rats more effectively than the known enzyme inhibitor, LJP-1207. Surprisingly, the SSAO activity was not increased, but decreased both in acute and chronic inflammation. Though experiments are in progress to clarify these findings, the enzyme might play a role in the very early phase of inflammation and be inactivated during leukocyte extravasation.

Protective effect of resveratrol against caspase 3 activation in primary mouse fibroblasts

Aim To study the effect of resveratrol on survival and caspase 3 activation in non-transformed cells after serum deprivation. Methods Apoptosis was induced by serum deprivation in primary mouse embryonic fibroblasts. Caspase 3 activation and lactate dehydrogenase release were assayed as cell viability measure by using their fluorogenic substrates. The involvement of PI3K, ERK, JNK, p38, and SIRT1 signaling pathways was also examined. Results Serum deprivation of primary fibroblasts induced significant activation of caspase 3 within 3 hours and reduced cell viability after 24 hours. Resveratrol dose-dependently prevented caspase activation and improved cell viability with 50% inhibitory concentration (IC50) = 66.3 ± 13.81 µM. It also reduced the already up-regulated caspase 3 activity when it was added to the cell culture medium after 3 hour serum deprivation, suggesting its rescue effect. Among the major signaling pathways, p38 kinase was critical for the protective effect of resveratrol which was abolished completely in the presence of p38 inhibitor. Conclusion Resveratrol showed protective effect against cell death in a rather high dose. Involvement of p38 kinase in this effect suggests the role of mild stress in its cytoprotective action. Furthermore due to its rescue effect, resveratrol may be used not only for prevention, but also treatment of age-related degenerative diseases, but in the higher dose than consumed in conventional diet.