Epäspesifisten ja spesifisten vuorovaikutuksien hyödyntäminen testosteronin ja sen sukuisten steroidien kapillaarielektromigraatioanalyyseissä

Determination of testosterone and related compounds in body fluids is of utmost importance in doping control and the diagnosis of many diseases. Capillary electromigration techniques are a relatively new approach for steroid research. Owing to their electrical neutrality, however, separation of steroids by capillary electromigration techniques requires the use of charged electrolyte additives that interact with the steroids either specifically or non-specifically. The analysis of testosterone and related steroids by non-specific micellar electrokinetic chromatography (MEKC) was investigated in this study. The partial filling (PF) technique was employed, being suitable for detection by both ultraviolet spectrophotometry (UV) and electrospray ionization mass spectrometry (ESI-MS). Efficient, quantitative PF-MEKC UV methods for steroid standards were developed through the use of optimized pseudostationary phases comprising surfactants and cyclodextrins. PF-MEKC UV proved to be a more sensitive, efficient and repeatable method for the steroids than PF-MEKC ESI-MS. It was discovered that in PF-MEKC analyses of electrically neutral steroids, ESI-MS interfacing sets significant limitations not only on the chemistry affecting the ionization and detection processes, but also on the separation. The new PF-MEKC UV method was successfully employed in the determination of testosterone in male urine samples after microscale immunoaffinity solid-phase extraction (IA-SPE). The IA-SPE method, relying on specific interactions between testosterone and a recombinant anti-testosterone Fab fragment, is the first such method described for testosterone. Finally, new data for interactions between steroids and human and bovine serum albumins were obtained through the use of affinity capillary electrophoresis. A new algorithm for the calculation of association constants between proteins and neutral ligands is introduced.Testosteronin ja sen sukuisten steroidien määrittäminen kehon nesteistä on keskeistä monien sairauksien diagnosoinnissa sekä doping-valvonnassa. Perinteiset steroidien analyysimenetelmät kärsivät usein joko riittämättömästä spesifisyydestä tai työläästä näytteiden esikäsittelystä. Tässä työssä tutkittiin testosteronin ja sen sukuisten steroidien määrittämistä kapillaarielektromigraatiotekniikoilla. Kyseiset tekniikat ovat varsin uusi lähtökohta steroiditutkimuksille. Kapillaarielektromigraatiotekniikat perustuvat molekyylien erottamiseen toisistaan voimakkaassa sähkökentässä. Nestemäinen näyte syötetään ohueen kapillaariin, jonka halkaisija on tyypillisesti 20 100 mikrometriä. Kapillaari on ennen näytteensyöttöä täytetty elektrolyyttinesteellä, ja näytteensyötön jälkeen sen molemmat päät asetetaan elektrolyyttiastioihin yhdessä elektrodien kanssa. Erotuksen aikaansaamiseksi elektrodien välille kytketään voimakas sähkökenttä. Sähköisesti varautuneet molekyylit (ionit) erottuvat sähkökentässä niiden erilaisten elektroforeettisten liikkuvuuksien mukaisesti. Steroidien sähköisestä varauksettomuudesta johtuen niillä ei ole elektroforeettista liikkuvuutta sähkökentässä. Kapillaarielektromigraatiotekniikoiden soveltaminen steroidien erottamiseen edellyttääkin ionisoituvien orgaanisten lisäaineiden käyttöä elektrolyyttiliuoksessa. Lisäaineilla voi olla joko spesifisiä tai epäspesifisia vuorovaikutuksia steroidien kanssa. Tässä työssä testosteronin ja sen sukuisten steroidien määrittämistä tutkittiin epäspesifisellä misellisellä sähkökineettisellä kromatografialla (MEKC). Elektrolyyttiliuoksessa käytettiin lisäaineina tensidejä sekä syklodekstriinejä. Kapillaari täytettiin vain osittain misellisellä elektrolyyttiliuoksella, minkä ansiosta steroidit voitiin tunnistaa sekä ultraviolettispektrofotometrisesti (UV) että sähkösumutus-ionisaatiomassaspektrofotometrisesti (ESI-MS). Osittaistäyttöön (PF) perustuva PF-MEKC UV osoittautui herkemmäksi, tehokkaammaksi ja toistettavammaksi menetelmäksi kuin PF-MEKC ESI-MS. Tutkimuksessa havaittiin, että ESI-MS-liitäntä asettaa huomattavia kemiallisia rajoituksia paitsi ionisaatio- ja detektointitapahtumille myös erotustapahtumalle. Uusilla PF-MEKC UV-menetelmillä steroidistandardit voidaan analysoida nopeasti, tehokkaasti ja kvantitatiivisesti. Herkin PF-MEKC UV-menetelmä soveltuu testosteronin nopeaan ja spesifiseen määrittämiseen miesten virtsanäytteistä miniatyrisoidun immunoaffiniteettikiinteäfaasiuuton (IA-SPE) jälkeen. Tutkimuksessa kehitetty IA-SPE perustuu vuorovaikutuksiin testosteronin ja sille spesifisen rekombinantin Fab-vasta-ainefragmentin välillä. IA-SPE on yksinkertainen ja nopea näytteen esikäsittelymenetelmä, jonka avulla tutkittava yhdiste voidaan konsentroida ja eristää muista näytemolekyyleistä. IA-SPE:tä ei ole aikaisemmin sovellettu testosteronin eristämiseen. Uutta tietoa steroidien sekä ihmisen ja naudan seerumin albumiiniproteiinien välisistä vuorovaikutuksista saatiin affiniteettikapillaarielektroforeesin avulla. Tutkimuksessa kehitettiin uusi algoritmi, jonka avulla voidaan laskea proteiinien ja neutraalien ligandien välisiä sitoutumisvakioita. Testosteronin ja sen sukuisten steroidien sitoutumisessa albumiiniproteiineihin havaittiin merkittäviä eroja

Steroid structural requirements for interaction of ostreolysin, a lipid-raft binding cytolysin, with lipid monolayers and bilayers

AbstractOstreolysin, a cytolytic protein from the edible oyster mushroom (Pleurotus ostreatus), recognizes and binds specifically to membrane domains enriched in cholesterol and sphingomyelin (or saturated phosphatidylcholine). These events, leading to permeabilization of the membrane, suggest that a cholesterol-rich liquid-ordered membrane phase, which is characteristic of lipid rafts, could be its possible binding site. In this work, we present effects of ostreolysin on membranes containing various steroids. Binding and membrane permeabilizing activity of ostreolysin was studied using lipid mono- and bilayers composed of sphingomyelin combined, in a 1/1 molar ratio, with natural and synthetic steroids (cholesterol, ergosterol, β-sitosterol, stigmasterol, lanosterol, 7-dehydrocholesterol, cholesteryl acetate, and 5-cholesten-3-one). Binding to membranes and lytic activity of the protein are both shown to be dependent on the intact sterol 3β-OH group, and are decreased by introducing additional double bonds and methylation of the steroid skeleton or C17-isooctyl chain. The activity of ostreolysin mainly correlates with the ability of the steroids to promote formation of liquid-ordered membrane domains, and is the highest with cholesterol-containing membranes. Furthermore, increasing the cholesterol concentration enhanced ostreolysin binding in a highly cooperative manner, suggesting that the membrane lateral distribution and accessibility of the sterols are crucial for the activity of this new member of cholesterol-dependent cytolysins

Albumin-hyaluronan interactions : influence of ionic composition probed by molecular dynamics

The lubrication mechanism in synovial fluid and joints is not yet fully understood. Nevertheless, intermolecular interactions between various neutral and ionic species including large macromolecular systems and simple inorganic ions are the key to understanding the excellent lubrication performance. An important tool for characterizing the intermolecular forces and their structural consequences is molecular dynamics. Albumin is one of the major components in synovial fluid. Its electrostatic properties, including the ability to form molecular complexes, are closely related to pH, solvation, and the presence of ions. In the context of synovial fluid, it is relevant to describe the possible interactions between albumin and hyaluronate, taking into account solution composition effects. In this study, the influence of Na+, Mg2+, and Ca2+ ions on human serum albumin–hyaluronan interactions were examined using molecular dynamics tools. It was established that the presence of divalent cations, and especially Ca2+, contributes mostly to the increase of the affinity between hyaluronan and albumin, which is associated with charge compensation in negatively charged hyaluronan and albumin. Furthermore, the most probable binding sites were structurally and energetically characterized. The indicated moieties exhibit a locally positive charge which enables hyaluronate binding (direct and water mediated)

Charakterizace interakcí barbiturátů a benzodiazepinů s lidskými sérovými proteiny pomocí kapilární elektroforézy

Důležitým směrem výzkumu při vývoji nových léčiv je jejich vazba na plasmatické proteiny. Tato diplomová práce charakterizuje interakce vybraných barbiturátů a benzodiazepinů s lidským sérovým albuminem a alfa glykoproteinem za fyziologických podmínek metodou kapilární elektroforézou - frontální analýzou. Navíc je sledována vazba vybraných barbiturátů a benzodiazepinů celkově ke všem proteinům plasmy kapilární elektroforézou po provedení ultrafiltrace. Výsledky ukazují, že rozhodující faktor pro vazbu na sérové proteiny je hydrofóbní charakter sloučenin.An important topic in the drug discovery and development process is the role of drug binding to plasma proteins. In this diploma thesis the characterization of the interaction between benzodiazepines and barbiturates towards human serum albumin and α-1-acid glycoprotein under physiological conditions by capillary electrophoresis-frontal analysis is presented. Furthermore, the binding of these drugs to all plasma proteins is evaluated by using ultrafiltration and capillary electrophoresis. The results indicate that the hydrophobic character of compounds seems to be the key factor on the interaction between these drugs towards proteins. In fact, hydrophobic basic drugs (benzodiazepines) bind in great extension to HSA, while less hydrophobic acid drugs (barbiturates) present lower interactions with proteins and bind especially to AGP.Katedra biochemických vědDepartment of Biochemical SciencesFaculty of Pharmacy in Hradec KrálovéFarmaceutická fakulta v Hradci Králov

Proteolytic Sensitivity, In Vitro Glycation Efficiency of Diabetic and Non-diabetic Human Serum Albumin

Background: Glycation of human serum albumin (HSA) leads to disturbances in its stability, activity, and other properties which, in turn, affect the functional properties of HSA. Modification of albumin by glycation shows considerable potential as a significant biochemical biomarker for diagnosing diabetes. The characteristics of the glycation process in proteins have not been fully examined yet and, therefore, there is insufficient knowledge about them in the field. Objectives: This study aimed to clarify the differences between diabetic and non-diabetic HSA as well as their structure-function relationship. Methods: The physiological and laboratory characteristics of glycated albumin as well as HSA were explored. A total of 30 subjects were enrolled in this study in which 15 normal healthy individuals were assigned into the control group, and 15 type-2 diabetic patients were included in the diabetic group. Patients with type-1 diabetes, pregnant women, and individuals with other diseases were excluded from the study. Protein estimation, polyacrylamide gel electrophoresis, ammonium sulphate fractionation, dialysis, glycation of HSA followed by gel electrophoresis of glycated samples, digestion of BSA, as well as HSA by α-chymotrypsin and their documentation and stoichiometry were all performed. Results: Various characteristic differences were observed between diabetic and non-diabetic HSA including proteolytic susceptibility and in vitro glycation efficiency. Hypoalbuminemia was, particularly, observed in diabetic patients, which was suggestive of a relationship between hyperglycemia and hypoalbuminemia. Conclusion: Peculiar contrariety between diabetic and non-diabetic HSA, specific differences in their glycation efficiencies, as well as proteolytic susceptibility and their innuendos were precisely traced out. It was concluded that albumin may have been regarded as a significant clinical biomarker for diagnosing diabetes

Steroid analysis by pH-mediated stacking MEKC

This dissertation is based on research that led to the development and application of capillary electrophoresis method for the analysis of sex steroid hormones in the blood or plasma of fish species. The analysis of steroid hormones is essential to reveal chemical compounds that are suspected in endocrine disruption, but it is challenging due to the similarity of the chemical structures of steroids, their low concentrations, and limited volumes of plasma or blood samples in fish available for analysis. There is therefore an acute need to develop reliable accurate and systematic analytical methods applicable for the analysis of structurally similar steroid hormones at very low concentrations. The method developed here is based on micellar electrokinetic chromatography, a type of capillary electrophoresis that incorporates secondary equilibria. The method utilizes pH-stacking for steroid preconcentration to improve the detection limits. This method of pH-stacking is accomplished by using charged derivatives of cyclodextrin which become neutral (protonated) or anionic (deprotonated) based on the pH of the sample buffer. Preconcentration by means of pH-stacking occurs upon introduction of the sample into the capillary at the pH junction, resulting in a fast and efficient separation analysis of steroids. Using the developed method the separation of eight targeted steroids, that include alpha,beta-dihydroxyprogesterone, ethynylestradiol, 17beta-estradiol, estrone, hydroxyprogesterone, 11-ketotestosterone, progesterone, and testosterone is achieved in less than 4 min which is substantially faster than steroid analysis by immunoassay, GC-MS or LC-MS. For all targeted steroids, the within-day and day-to-day reproducibility in migration time is \u3c1 and \u3c2% relative standard deviation (RSD), respectively. The reproducibility in peak area obtained in aqueous samples is below 6% and 22% (RSD) within-day and day-to-day respectively. The limits of detection range from 2 to 14 nM using a 60 s electrokinetic injection. The method is validated by measuring the recovery of standard steroids added to the aqueous or fish plasma samples prior to sample preparation. The recovery of testosterone and 17beta-estradiol added to the fish plasma prior to sample preparation range from 74% to 102%. The method is successfully applied to the determination of sex steroid levels in blood plasma of yellow perch captured from natural aqueous habitats. The results are compared to radioimmunoassay