Četvrt veka iskustva u redoks biohemiji: razotkrivanje uloge oksidativnog stresa u zdravlju i bolesti

Oxidative stress is a pathphysiological phenomenon that occurs during various physiological processes, as a consequence of aging, but also in many diseases. Oxidative stress "eruption" occurs when its factors - free radicals are increased in pathological conditions, when their creation overcomes the mechanisms of antioxidant protection. Measurement of redox status parameters gives new biomarkers of various diseases. During more than 25 years of dealing with this topic, we analyzed the redox status of a large number of patients with various diseases: cardiovascular, stroke, kidney disease, diabetes, cancer, obesity. Redox status was determined in a large number of healthy subjects, and in special physiological conditions: pregnancy, athletes, children, elderly, in subjects after antioxidants' supplementation. Oxidative stress markers measurement is applied in pharmacological studies on several different animal models: atherosclerosis, multiple sclerosis, blast injuries, poisoning, rheumatoid arthritis. These types of studies provide insight into cellular oxidative stress development, mechanisms involved in the disease development, insight into the possibility of its modulation under the therapy influence. Many years of experience in the oxidative stress analysis, enabled us to develop a system for testing the antioxidant potential of biologically active compounds in the serum pool of healthy individuals. The redox status of the respondents is "chemical ID card", which changes in various diseases. "Redox imbalance" diagnosis would allow determining the type of antioxidants' application, dosing regimen, monitoring the therapy effects, which is the application of the principles of precision (personalized) medicine, as the ideal of modern therapy.Oksidativni stres je patofiziološki fenomen koji se javlja tokom različitih fizioloških procesa, kao posledica starenja, ali i u mnogim bolestima. “Erupcija” oksidativnog stresa nastaje kada se njegovi činioci tzv. slobodni radikali povećano stvaraju u određenim patološkim stanjima, kada njihovo stvaranje nadvlada mehanizme i činioce antioksidativne zaštite. Merenje parametara redoks statusa daje nove biomarkere različitih bolesti . Tokom više od 25 godina bavljenja ovom temom analizirali smo redoks status velikog broja pacijenata sa različitim bolestima: kardiovaskularnim, moždanim udarom, akutnim i hroničnim bubrežnim bolestima, dijabetesom, kancerom, gojaznošću. Redoks status je određivan i kod velikog broja zdravih ispitanika, kao i u posebnim fiziološkim stanjima: trudnice, sportisti, deca, stare osobe, kao i kod ispitanika nakon suplementacije antioksidansima. Merenje markera oksidativnog stresa je primenjeno i u farmakološkim studijama na više različitih animalnih modela: ateroskleroza, multipla skleroza, blast povreda, trovanja, reumatoidni artritis. Ovakve vrste studija omogućavaju uvid u razvoj ćelijskog oksidativnog stresa, mehanizme koji su uključeni u razvoj bolesti, kao i uvid u mogućnosti njegove modulacije pod uticajem terapije. Dugogodišnje iskustvo u analitici oksidativnog stresa nam je omogućilo razvoj sistema za testiranje antioksidativnog potencijala biološki aktivnih jedinjenja u serumskom pulu zdravih osoba. Rеdoks status ispitanika je njegova “hеmijska lična karta”, koja se menja u različitim stanjima i bolestima. Dijagnoza “rеdoks disbalansa“ kod odrеđеnе osobе, omogućila bi odrеđivanjе vrstе i načina primеnе antioksidanasa, rеžim doziranja, praćеnjе еfеkata primеnе, što bi predstavljalo primenu principa prеciznе (pеrsonalizovanе) medicine, kao ideala moderne terapije.VIII Kongres farmaceuta Srbije sa međunarodnim učešćem, 12-15.10.2022. Beogra

Electrochemical Characterisation and Confirmation of Antioxidative Properties of Ivermectin in Biological Medium

Ivermectin (IVM) is a drug from the group of anthelmintics used in veterinary and human medicine. Recently, interest in IVM has increased as it has been used for the treatment of some malignant diseases, as well as viral infections caused by the Zika virus, HIV-1 and SARS-CoV-2. The electrochemical behaviour of IVM was investigated using cyclic (CV), differential pulse (DPV) and square wave voltammetry (SWV) at glassy carbon electrode (GCE). IVM showed independent oxidation and reduction processes. The effect of pH and scan rate indicated the irreversibility of all processes and confirmed the diffusion character of oxidation and reduction as an adsorption-controlled process. Mechanisms for IVM oxidation at the tetrahydrofuran ring and reduction of the 1,4-diene structure in the IVM molecule are proposed. The redox behaviour of IVM in a biological matrix (human serum pool) showed a pronounced antioxidant potential similar to that of Trolox during short incubation, whereas a prolonged stay among biomolecules and in the presence of an exogenous pro-oxidant (tert-butyl hydroperoxide, TBH) resulted in a loss of its antioxidant effect. The antioxidant potential of IVM was confirmed by voltametric methodology which is proposed for the first time

Oxidative stress and cardiometabolic biomarkers in patients with non-alcoholic fatty liver disease

Oxidative stress is assumed to be the underlying feature of non-alcoholic fatty liver disease (NAFLD). To our knowledge, the mutual involvement of redox status homeostasis parameters [i.e., advanced oxidation protein products (AOPP), pro-oxidant-antioxidant balance (PAB), total oxidant status (TOS), total antioxidant status (TAS) and oxidative-stress index (OSI)] and cardiometabolic biomarkers in subjects with NAFLD has not been examined yet. Accordingly, we aimed to investigate this potential relationship. A total of 122 subjects with NAFLD were compared with 56 participants without NAFLD. The diagnosis of NAFLD was confirmed by abdominal ultrasound. Anthropometric and biochemical parameters were measured. OSI, Castelli’s Risk Index I (CRI-I) and Castelli’s Risk Index II (CRI-II) were calculated. Univariate and multivariate binary logistic regression analysis were used to test the predictions of oxidative stress and cardiometabolic markers, respectively for NAFLD. Principal component analysis (PCA) was applied to explore its mutual effect on NAFLD status. Significant positive associations of CRI-I, CRI-II, high sensitivity C-reactive protein (hsCRP) and AOPP with NAFLD were found. PCA analysis extracted 3 significant factors: Oxidative stress-cardiometabolic related factor (i.e., triglycerides, AOPP, HDL-c and HbA1c)-explained 36% of variance; Pro-oxidants related factor (i.e., TOS and PAB)-explained 17% of variance; and Antioxidants related factor (i.e., TAS)- explained 15% of variance of the tested parameters. Moreover, binary logistic regression analysis revealed significant predictive ability of Oxidative stress-cardiometabolic related factor (p < 0.001) and Pro-oxidants related factor (p < 0.05) for NAFLD status. In addition to oxidative stress (i.e., determined by higher AOPP levels), dyslipidemia (i.e., determined by higher lipid indexes: CRI-I and CRI-II) and inflammation (determined by higher hsCRP) are independently related to NAFLD status. The mutual involvement of pro-oxidants (i.e., TOS and PAB), or the joint involvement of pro-oxidants (i.e., AOPP) and cardiometabolic parameters (i.e., HbA1c, triglycerides and HDL-c) can differentiate subjects with NAFLD from those individuals without this metabolic disorder. New studies are needed to validate our results in order to find the best therapeutic approach for NAFLD

Primena LC‐MS/MS metoda u ispitivanju parametara oksidativnog stresa

Oxidative stress is a phenomenon that occurs due to the disturbance in the balance between the production of reactive oxygen species and the ability of biological systems to remove the resulting compounds. Oxidative stress is involved in the pathogenesis of many disorders in the human organism. This indicates the importance of quantification of oxidative stress parameters in biological samples. Traditionally, these parameters are determined by biochemical tests. Although these tests are routinely performed, they have many drawbacks. To determine the exact concentration of selected compounds, more sensitive analytical methods are becoming more important. In the modern scientific literature, liquid chromatography-tandem mass spectrometry (LC-MS/MS) is increasingly mentioned (1). This method, with adequate selection of stationary and mobile phases, enables quantification of very low concentrations of selected parameters. In addition, these methods can simultaneously determine the concentration of many selected components. However, it is necessary to take into account that LC-MS/MS methods require a very purified biological sample from which the proteins have been maximally removed. In this work, we will present the results of determination of cysteine, cystine, reduced and oxidized glutathione in patients with microcellular lung cancer. The use of LC-MS/MS methods is becoming increasingly common for the analysis of oxidative stress markers in biological fluids. In the future, we are expected to move to even more modern, fully automated methods, which simultaneously purify and analyze samples.Oksidativni stres je pojava koja nastaje usled narušavanja ravnoteže između proizvodnje i nagomilavanja reaktivnih jedinjenja kiseonika u organizmu i sposobnosti bioloških sistema da ukloni nastala jedinjenja. Oksidativni stres učestvuje u patogenezi mnogih poremećaja kao što su kardiovaskularne bolesti, dijabetes i bolesti bubrega. Ovo ukazuje na značaj određivanja odabranih parametara oksidativnog stresa u uzorcima biološkog materijala. Tradicionalno, najveći broj parametara se određuje biohemijskim testovima. Iako se ovi testovi rutinski izvode, oni imaju mnoge nedostatke. Da bi se odredila tačna koncentracija izabranih parametara, koji se u uzorcima nalaze u veoma niskim koncentracijama, sve veću prednost imaju osetljivije analitičke metode. U savremenoj naučnoj literaturi se u te svrhe sve više spominje tečna hromatografija spregnuta sa masenim detektorom (LC-MS/MS) (1). Ova metoda, uz adekvatan izbor stacionarne i mobilne faze, omogućava kvantifikaciju veoma niskih koncentracija odabranih jedinjenja. Sem toga, ovim metodama se istovremeno, u jednom uzorku, može odrediti koncentracija većeg broja odabranih komponenti. Ipak, neophodno je voditi računa o tome da tečna hromatografija spregnuta sa masenim detektorom zahteva dobro prečišćen uzorak biološkog materijala iz kog su maksimalno uklonjeni proteini. U ovom radu biće prikazani rezultati određivanja cisteina, cistina, redukovanog i oksidovanog glutationa kod pacijenata sa mikrocelularnim karcinomom pluća. Upotreba LC-MS/MS metoda je sve uobičajenija za analizu markera oksidativnog stresa u biološkim tečnostima zbog svoje osetljivosti. U budućnosti se očekuje razvoj ka još savremenijim, potpuno automatizovanim metodama, kojima se istovremeno uzorci prečišćavaju i analiziraju.VIII Kongres farmaceuta Srbije sa međunarodnim učešćem, 12-15.10.2022. Beogra

Redox homeostasis, oxidative stress and antioxidant system in health and disease: The possibility of modulation by antioxidants

Redox imbalance occurs when the factors of oxidative stress, known as prooxidants, outweigh the mechanisms of antioxidant protection. In a healthy state, homeostatic mechanisms ensure the balanced production of free radicals and a complete series of antioxidants responsible for their safe removal. The generation of free radicals is a part of physiological processes in a healthy organism, some of which act as specific signaling molecules, and their presence and activity are necessary in these processes. In various diseases such as cancer, cardiovascular disease, diabetes, autoimmune diseases, rheumatic diseases, systemic lupus, and skin diseases, the generation of free radicals overwhelms the protective mechanisms, leading to the development of "oxidative stress" that damages cells and tissues. To prevent the harmful effects of free radicals within cells, there exists a system of enzymatic antioxidant protection composed of superoxide dismutase (SOD), glutathione peroxidase (GSHPx), glutathione reductase (GR), glutaredoxin, reduced/oxidized glutathione (GSH/GSSG), and thioredoxin (TRX). The examples of non-enzymatic antioxidants are: antioxidant vitamins such as A, C and E, dihydrolypoic acid, metallothioneins, ceruloplasmin, coenzyme Q 10, urea, creatinine, etc. Redox balance is influenced by the circadian rhythm and external factors that constitute the "exposome", including dietary habits and lifestyle. Antioxidant supplementation has become increasingly popular for maintaining optimal body function. However, it is important to note that some antioxidants can exhibit prooxidant activity, emphasizing the need for controlled use. The relationship between the redox status of the body and the action of antioxidants enables the development of multidisciplinary research that connects biochemistry, molecular biology, nutritional science, natural product chemistry, and clinical practice

Malnutricija uzrokovana karcinomom i onkološkom terapijom

Malnutrition as a consequence of the disease occurs most often in cancer patients, not only because of the cancer itself, but also because of the oncological treatment. The most important cause is chemotherapy, followed by ra- diotherapy and onco-surgery. Therefore, efforts are being made all over the world to find different ways to solve this difficult medical problem. Given that the mechanism of malnutrition caused by cancer is not sufficiently elucidat- ed, the efforts made to design an adequate approach and treatment of this disorder are often unsuccessful. In order to improve the nutritional status of oncology patients, their nutritional disorder should be adequately diagnosed and then specific measurements should be performed (deter- mination of body composition, the proportion of water, fat, muscle, as well as the determination of various biomarkers in the blood, for example markers inflammation). In this way, it is possible to formulate a multimodal approach to the treatment of malnutrition in oncology patients, which would include the intake of adequate foods, increased physical activity and personalized supplementation.Pothranjenost kao posledica bolesti se najčešće javlja kod onkoloških pacijenata i to ne samo zbog samog kancera, već i zbog onkološkog lečenja. Najznačajniji uzročnik je hemioterapija, potom radioterapija i onko-hirurgija. Zbog toga se širom sveta ulažu napori i pronalaze različiti načini za rešavanje ovog teškog medicinskog problema. S obzirom na to da mehanizam nastanka pothranjenosti uzrokovane karcinomom nije dovoljno rasvetljen, napori koji se ulažu da se osmisli adekvatan pristup i tretman ovog poremećaja se često neuspešno završe. Da bi se nutritivni status onko- loških pacijenata popravio njima pre svega treba da bude dijagnostikovan nutritivni poremećaj, a zatim da se sprovedu specifična merenja (određivanje sastava tela, udeo vode, masti, mišića, kao i da se u krvi odrede različiti biomarkeri, na primer markeri zapaljenja). Na taj način je moguće formulisati multimodalni pristup u lečenju pothranjenosti onkoloških bolesnika koji bi obuhvatio unos adekvatnih namirnica, povećanje fizičke aktivnosti i personalizovanu suplementaciju

In vitro antioxidant activity of Filipendula ulmaria (L.) Maxim. and Filipendula vulgaris Moench

Filipendula ulmaria (L.) Maxim. and F. vulgaris Moench are traditionally employed in the treatment of peptic ulcer and pain. The aim of present study was to estimate their antioxidant properties in order to better understand reported ethnomedicinal use. Antioxidant activity of lyophilized flower infusions (LFIs), spiraeoside (quantitatively dominant flavonoid) and referent compound Trolox® was investigated in human serum in vitro by following their ability to decrease oxidative stress produced by the addition of tert-butyl hydroperoxide. Parameters indicating protective capacity (total antioxidative status, TAS; total oxidative status, TOS; and pro-oxidant–antioxidant balance, PAB) were determined spectrophotometrically and results were expressed as percentage of the value of the same parameter in control group. All tested samples (10–160 μg/mL) concentration-dependently and significantly increased TAS (for maximum concentration, values were 190-400%) and decreased PAB (for maximum concentration, values were 13-78%). LFIs in the applied concentration range did not significantly influence TOS, in contrast to spiraeoside which increased its values, and Trolox® which reduced TOS. Protective activity of Trolox® was more pronounced than the effect of herbal preparations and spiraeoside. The obtained results support folkloric use of F. ulmaria and F. vulgaris and indicate considerable antioxidant capacity which should be further investigated.Saopštenje sa međunarodnog skupa štampano u izvodu (M34

Socio-demographic factors associated with smoking habits among university students in Belgrade, Serbia

Background: Smoking rates in Serbian adults are among the highest in Europe. The objective of this study is to assess the prevalence of smoking and smoking-related behaviours of Belgrade University students depending on their sociodemographic characteristics and faculty group. Methods: A cross-sectional study was carried out among 2,608 Belgrade University students (59.6% female) in 2015. A self-administered questionnaire was applied to the opportunity sample to collect the data describing students' smoking habits and attitudes across all 30 faculties of the university. Results: 30.5% of students reported smoking: 26.4% of medical, and 31.1% of non-medical ones. Smoking rate among female students was 31.2% vs. 29.5% among males. Age (p=0.001), relationship ( lt 0.001) and employment status (p=0.002) had statistically significant influence on smoking status, while the differences in smoking status between genders (p=0.141) and medical and non-medical group of students (p=0.066) were not statistically significant. The highest percentage of students started smoking during high school (66.2%). As the most common reason to start smoking, respondents cited peer influence (36.5%). 44.3% of students who smoked unsuccessfully tried to quit smoking. Conclusion: To combat high smoking prevalence among a younger population, the formal education of students about the adverse impacts of smoking should be integrated in all active anti-smoking programs. Medical students, as future healthcare professionals, can play an important role in smoking rates reduction among both younger and general populations, if properly trained and educated about smoking prevention and cessation techniques

Antioksidativna svojstva lipoproteina visoke gustine: više od kardiovaskularne zaštite

High-density lipoprotein (HDL) is the most complex lipoprotein particle, containing lipids and dozens of various functional proteins. Such sophisticated composition enables numerous activities of HDL; from the reverse cholesterol transport, to antioxidative, anti- inflammatory, anti-aggregation, antiadhesive, and vasodilatory effects. Accordingly, the significance of HDL goes far beyond its cardioprotective properties and novel research points towards its role in etiopathogenesis of various other diseases. Antioxidative properties of HDL are primarily attributed to the enzyme paraoxonase 1 (PON1), whose principal role is to protect low-density lipoprotein (LDL) and cell membranes against harmful effects of reactive oxygen species (1). PON1 is located on HDL particles and its activity largely depends on HDL structure. Our investigations have shown that PON1 is not equally distributed across the entire population of serum HDL subfractions. Namely, our results suggest that the allocation of PON1 on specific HDL subclasses changes in pathophysiological conditions, such as chronic kidney disease, polycystic ovary syndrome, or hypertension in pregnancy. Similarly, a shift in HDL subclasses distribution toward smaller, dysfunctional particles is reported in diabetes, metabolic syndrome, obstructive sleep apnea, sarcoidosis, but also in malignant diseases, such as colorectal cancer. In parallel, antioxidative defense mechanisms were diminished in all these categories of patients, which was evident as decreased PON1 activity and rise of oxidative stress. Structural modifications of HDL particles affect their functions, thus antioxidative capability of PON1 depends on qualitative properties of its lipoprotein carrier. Such complex interaction is highly significant for the initiation and progression of numerous diseases.Lipoprotein visoke gustine (high‐density lipoprotein, HDL) je najkompleksnija lipoproteinska čestica koja, pored lipidnih komponenti, sadrži i desetine različitih funkcionalnih proteina. Ovakav složen sastav omogućava brojne funkcije HDL; od reverznog transporta holesterola, do antioksidativnih, antiinflamatornih, antiagregacijskih, antiadhezivnih i vazodilatatornih svojstava. U skladu s tim, značaj HDL u mnogome prevazilazi kardioprotektivne efekte, a savremena otkrića ukazuju na ulogu ovih čestica u etiopatogenezi različitih oboljenja. Antioksidativna svojstva HDL prevashodno su vezana za enzim paraoksonazu 1 (PON1), čija je osnovna funkcija zaštita lipoproteina niske gustine (low‐density lipoprotein, LDL) i ćelijskih membrana od oksidativnih oštećenja (1). PON1 je locirana na HDL česticama i uočeno je da aktivnost ovog enzima u velikoj meri zavisi od strukture samog HDL. U našim istraživanjima pokazali smo da PON1 nije ravnomerno zastupljena na svim subfrakcijama HDL, te da se ova zastupljenost menja u različitim patofiziološkim stanjima, kao što su hronične bubrežne bolesti, sindrom policističnih jajnika, ili hipertenzija u trudnoći. Slično tome, pomeranje raspodele HDL subfrakcija ka manjim, disfunkcionalnim česticama uočeno je kod pedijatrijskih i odraslih pacijenata sa dijabetesom, metaboličkim sindromom, opstruktivnom apnejom u toku spavanja, sarkoidozom, ali i sa malignim bolestima, kao što je kolorektalni karcinom. Istovremeno, nivo antioksidativne zaštite kod ovih pacijenata je bio snižen, što je bilo vidljivo i kao smanjenje aktivnosti PON1, te sledstveno povećanje nivoa oksidativnog stresa. Strukturne modifikacije HDL čestica utiču na njihovu funkciju, pa tako i antioksidativni kapacitet enzima PON1 zavisi od kvalitativnih svojstava njegovog lipoproteinskog nosača. Ovakva složena interakcija između HDL i pridruženih funkcionalnih proteina značajna je za nastanak i progresiju brojnih oboljenja.VIII Kongres farmaceuta Srbije sa međunarodnim učešćem, 12-15.10.2022. Beogra

Ex vivo and in vivo antioxidant activity of β-hydroxy-β-arylalkanoic acids

The interplay between oxidative stress and inflammation is implicated in many chronic diseases including Alzheimer`s disease, cardiovascular diseases, diabetes and cancer. Thirteen β-hydroxy-β-arylalkanoic acids were previously synthesized and evaluated for their anti-inflammatory activity. The aim of this study was to asses ex vivo antioxidant activity of synthesized acids, as well as ibuprofen and to identify the compounds with the most promising results for further investigation on their capacity to counteract in vivo oxidative stress triggered by inflammation. The antioxidant potential of tested compounds was evaluated by determining the concentrations of total antioxidative status, total oxidative status, prooxidant antioxidant balance and the total sulfhydryl groups. Z score statistics were used to calculate the summary scores for antioxidative activity, prooxidative activity and oxy score. The tested compounds and ibuprofen demonstrated mild prooxidative activity ex vivo. Seven acids with substituents on one benzene ring exhibited better results than ibuprofen and were selected for in vivo testing. In vivo results demonstrated better antioxidant protection compared to ex vivo results. Compound g which contains nitro group on the benzene ring demonstrated the lowest oxy score, and four compounds exhibited better results than ibuprofen