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Predmet istraživanja ove doktorske teze je in vitro određivanje antiradikalske aktivnosti odabranih neurotranmitera, njihovih prekursora i metabolita, a u cilju uspostavljanja odnosa na liniji struktura-antiradikalska aktivnost. Rezultati su upotpunjeni teorijskim proračunima kako bi se dobila potpuna slika parametara važnih za aktivnost prema radikalima od interesa, ali odredili termodinamički i kinetički najpovoljniji mehanizmi. Veliki broj spoljašnjih faktora utiče na povećanje koncentracije slobodnih radikala u organizmu pri čemu nastaje oksidacioni stres. U ovom procesu se ireverzibilno oštećuju biološki značajni molekuli i to dejstvom reaktivnih vrsta kiseonika, azota, hlora i sumpora. U literaturi je pokazano da su slobodni radikali direktno ili indirektno povezani sa razvojem neurodegenerativnih bolesti (Parkinsonove, Alchajmerove, šizofrenije i amiotrofične lateralne skleroze). Aktivnost može biti izražena ili kroz oksidaciju biološki molekula ili formiranje polimernih struktura, odnosno plakova. Većina ispitivanih egzogenih antioksidanasa ne može preći krvno-moždanu barijeru, tako da se ne mogu direktno koristiti za lečenje neurodegenerativnih bolesti. Zbog toga se sve veći značaj pridaje molekulima koji su prisutni u organizmu. Po svojoj strukturi, većina neurotransmitera pripada klasi kateholamina, odnosno poseduju kateholnu grupu koja je i čest strukturni parametar dobrih antioksidanasa. Dopamin, norepinefrin, epinefrin, njihov prekursor L-DOPA i dopaminski metabolit DOPAC poseduju ovu strukturnu jedinicu. Zamenom jedne hidroksilne grupe metoksigrupom nastaju 3-metoksitiramin, homovanilinska kiselina, vanililbademova kiselina (vanillylmandelic acid – VMA), značajni metaboliti neurotransmitera koji se eksperimentalno određuju prilikom dijagnoze bolesti povezanih sa promenom koncentracije dopamina. Tiramin, tirozin i oktopamin imaju hidroksilnu grupu u ppoložaju u odnosu na alifatični niz dok fenilalanin i feniletilamin u strukturi nemaju hidroksilne grup, The research topic of this doctoral dissertation is in vitro determination of antiradical activity of selected neurotransmitters, their precursors and metabolites, with the main goal of establishment of the structure-antiradical activity relationship. The results incorporate theoretical calculations in order to obtain a full picture of parameters important for activity towards radicals of interest, but also to determine the thermodynamically and kinetically most favorable mechanism. A large number of external factors influences the increase in the concentration of free radicals which leads to oxidative stress. In this process, the biologically important molecules are irreversibly damaged by the activity of reactive oxygen, nitrogen, chlorine and sulfur species. It is shown in the literature that free radicals are directly or indirectly connected to the development of neurodegenerative diseases (Parkinson, Alzheimer, schizophrenia, amyotrophic lateral sclerosis). The activity of the reactive species is expressed through oxidation of biologically important molecules or the formation of polymeric structures, plaque. The most of investigated exogenous antioxidants cannot cross the blood-brain barrier, therefore they cannot be used for the treatment of neurodegenerative diseases. Because of this, there is an increased interest in the naturally occurring molecules in an organism. Structurally, the majority of neurotransmitters are catecholamines, which means that they possess catechol moiety, the common structural motif of good antioxidants. Dopamine, norepinephrine, epinephrine, their precursor L-DOPA and dopamine metabolite DOPAC have this group. When one of the hydroxyl groups is exchanged with methoxy group the following molecules are obtained: 3-methoxytyramine, homovanillic acid and vanillylmandelic acid. These molecules are experimentally determined in the diagnosis of diseases resulting from a change in the dopamine concentration. Tyramine, tyrosine, and octopamin

The delicate balance between the advantageous and detrimental effects of free radicals is one of the important aspects of human (patho)physiology. The controlled production of reactive oxygen and nitrogen species has an essential role in the regulation of various signaling switches. On the other hand, imbalanced generation of radicals is highly correlated with the pathogenesis of many diseases which require the application of selected antioxidants to regain the homeostasis. In the era of growing interest for redox processes, electron paramagnetic resonance (EPR) spectroscopy is arguably the best-suited technique for such research due to its ability to provide a unique insight into the world of free radicals and antioxidants. Herein, I present the principles of EPR spectroscopy and the applications of this method in assessing: (i) the oxidative status of biological systems, using endogenous long-lived free radicals (ascorbyl radical (Asc(center dot)), tocopheroxyl radical (TO center dot), melanin) as markers; (ii) the production of short-lived radicals (hydroxyl radical (OH center dot), superoxide radical anion (O-2(-)), sulfur-and carbon-centered radicals), which are implicated in both, oxidative stress and redox signaling; (iii) the metabolism of nitric oxide (NO center dot); (iv) the antioxidative properties of various drugs, compounds, and natural products; (v) other redox-relevant parameter. Besides giving a comprehensive survey of up-to-date literature, I also provide illustrative examples in sufficient detail to provide a means to exploit the potential of EPR in biochemical/physiological/medical research. The emphasis is on the features and characteristics (both positive and negative) relevant for EPR application in clinical sciences. My aim is to encourage fellow colleagues interested in free radicals and antioxidants to expand their base knowledge or methods used in their laboratories with data acquired by EPR or some of the EPR techniques outlined in this r

Under physiological conditions oxygen is constantly being converted to reactive oxygen intermediates, in mitochondria, peroxisomes, cytochrome p450 systems, macrophages, neutrophils and in plasma membranes. These reactive oxygen species (ROS) are toxic and therefore alter cell integrity leading to cell damage. To protect itself against this toxic effect of ROS, living systems have developed defence systems that scavenge ROS formation. These systems include some enzymes, transporting proteins and small antioxidant molecules for instance vitamin C and E. This thesis describes a study on the antioxidant chemistry and activity of vitamin C in vivo and in vitro systems using ESR spectroscopy. Also, a new method was designed to label ascorbic acid with a fluorescent marker. Moreover, some important criteria were considered for the evaluation and quantification of ascorbyl radicals in human blood plasma using two types of ESR spectrometers., In-vivo- und in-vitro-Untersuchungen über hoch dosiertes und Fluoreszenz-markiertes Vitamin C mit ESR- und Fluoreszenzspektroskopie

Under physiological conditions oxygen is constantly being converted to reactive oxygen intermediates, in mitochondria, peroxisomes, cytochrome p450 systems, macrophages, neutrophils and in plasma membranes. These reactive oxygen species (ROS) are toxic and therefore alter cell integrity leading to cell damage. To protect itself against this toxic effect of ROS, living systems have developed defence systems that scavenge ROS formation. These systems include some enzymes, transporting proteins and small antioxidant molecules for instance vitamin C and E. This thesis describes a study on the antioxidant chemistry and activity of vitamin C in vivo and in vitro systems using ESR spectroscopy. Also, a new method was designed to label ascorbic acid with a fluorescent marker. Moreover, some important criteria were considered for the evaluation and quantification of ascorbyl radicals in human blood plasma using two types of ESR spectrometers., In-vivo- und in-vitro-Untersuchungen über hoch dosiertes und Fluoreszenz-markiertes Vitamin C mit ESR- und Fluoreszenzspektroskopie