Hrana je glavni izvor izloženosti ljudi esencijalnim i toksičnim metalima, uključujući kadmij (Cd). Cilj istraživanja je bio procijeniti učinke peroralne izloženosti Cd tijekom skotnosti štakorice na ...razdiobu kadmija i stanje mikronutrijenata u majčinom organizmu i fetusu te funkcije posteljice u prijenosu nutrijenata i sintezi steroidnih hormona. Hipoteze istraživanja su bile da se tijekom graviditeta povećava želučanocrijevna apsorpcija Cd koji se nakuplja u posteljici i može poremetiti prijenos nutrijenata do fetusa i sintezu posteljičnih hormona. Pokusne štakorice (Wistar) su nakon parenja izlagane dozi od 50 mg Cd/l (u obliku CdCl2xH2O) u vodi za piće od 1. do 19. ili 20. dana skotnosti. Neskotne štakorice su istodobno izlagane pod jednakim uvjetima izloženosti tijekom 20 dana. Posljednjeg dana pokusa je svim štakoricama u općoj anesteziji izvađena krv iz srca i uzorkovani unutrašnji organi, posteljice i fetusi, koji su pripremljeni za analize mikroelemenata (metodom AAS). Progesteron i testosteron su analizirani imunokemijski izravno u serumu (metodom IEMA) i u uzorcima pripremljenim iz posteljičnog tkiva (metodama IEMA i/ili ELISA). U svih izloženih štakorica su nađena povećanja Cd u svim izmjerenim uzorcima i cinka u jetri. Količine željeza u fetusu i cinka u fetusu i/ili posteljici su bile smanjene. U izloženih skotnih vs. izložene neskotne štakorice su bila izraženija povećanja koncentracije Cd u krvi i količina Cd u jetri i bubregu kao i smanjenja količina željeza u jetri i bubregu te cinka i bakra u bubregu. U svih skotnih vs. neskotne štakorice su bile veće koncentracije Cd u krvi i količine bakra u bubregu te manje količine željeza u jetri i bubregu i bakra u jetri. Nije bilo promjena u steroidnim hormonima ni u serumu, ni u posteljici, u kojoj su vrijednosti svakog hormona izmjerene dvjema imunokemijskim metodama značajno korelirale. U zaključku, ovim radom su dobiveni izvorni podaci o vrijednostima progesterona i testosterona (kao prekusora za sintezu estradiola u jajniku) u serumu i posteljici štakorice blizu roka okoćenja. Novi i izvorni znanstveni rezultati su da peroralna izloženost štakorica dozi od 6,5 mg Cd/kg tjelesne mase otopinom za napajanje tijekom skotnosti povećava razine Cd u krvi, jetri i bubregu s posljedičnim biokemijskim promjenama mikronutrijenata u većoj mjeri nego u neskotnih štakorica što istodobno s nakupljanjem Cd u posteljici remeti transplacentarni prijenos željeza i cinka te može predstavljati opasnost za rast i razvoj fetusa in utero.
Food is the main source of human exposure to essential and toxic metals, including cadmium (Cd). This investigation aimed to assess the effects of oral Cd exposure during rat pregnancy on Cd distribution and micronutrient status in a maternal organism and foetus and placental functions in nutrient transport and steroid hormone synthesis. Research hypotheses were that Cd gastrointestinal absorption increases during pregnancy and Cd accumulates in the placenta where it may interfere with nutrient transport to the foetus and placental hormone biosynthesis. Female rats (Wistar) were mated and exposed to 50 mg Cd/l (as CdCl2xH2O) in drinking water from gestation day 1 through 19 or 20. Non-pregnant rats were concurrently exposed during 20 days under the same exposure conditions. On the last experimental day, under general anaesthesia, blood was taken by cardiac puncture from all of the rats and internal organs, placentas and foetuses were dissected and prepared for element analysis (by AAS). Progesterone and testosterone were assayed by immunochemical methods directly in sera (by IEMA) and in placental tissue-derived samples (by IEMA and/or ELISA). All of the exposed rats exhibited increases in Cd in all of the analysed samples and zinc in the liver. Contents of iron in the foetus and zinc in the foetus and/or placenta were decreased. In exposed pregnant vs. exposed non-pregnant rats, more pronounced increases in Cd concentration in the blood and Cd contents in the liver and kidney, decreases in iron contents in the liver and kidney, and decreases in zinc and copper contents in the kidney were recorded. In all pregnant vs. non-pregnant rats, higher Cd concentrations in the blood and copper content in the kidney and lower iron contents in the liver and kidney and copper content in the liver were found. Steroid hormones did not change in either the serum or placenta; in the latter the values of either hormone measured by two immunochemical assays were correlated. In conclusion, this work provides original evidence on progesterone and testosterone (as the precursor for the ovarian oestradiol synthesis) in rat serum and placenta at term. New and original research results are that oral Cd exposure to 6.5 mg Cd/kg body mass in drink during rat pregnancy increases levels of Cd in the blood, liver and kidney with consequent biochemical changes of micronutrients more pronouncedly than in non-pregnant rats, which together with accumulation of Cd in the placenta disrupts the transplacental handover of iron and zinc and may put at risk foetal growth and development in utero.
Uvod: Glavni regulator sistemske homeostaze željeza je hepcidin, čiju sintezu reguliraju status željeza, upalni citokini, eritropoetska aktivnost, hipoksija i anemija. Cilj ovoga rada bio je ispitati ...povezanost između serumskog hepcidina i čimbenika koji ga reguliraju u skupini bolesnika s KOPB-om, da bi se ispitalo kako ovi čimbenici utječu na koncentraciju hepcidina i osiguravanje opskrbe željezom neophodnim za eritropoezu. Hipoteze istraživanja: koncentracija hepcidina u serumu bolesnika s KOPB-om promijenjena je u odnosu na kontrolnu skupinu i mijenja se tijekom egzacerbacije u odnosu na stabilnu fazu bolesti; na koncentraciju hepcidina u KOPB-u utječu upala i/ili hipoksija.
Metode i ispitanici: U istraživanje je bilo uključeno 40 bolesnika s KOPB-om i 30 zdravih ispitanika. U KOPB-u skupini parametri su longitudinalno praćeni u tri vremenske točke: u egzacerbaciji, fazi rezolucije i stabilnoj fazi bolesti. Svim ispitanicima određena je koncentracija hepcidina, pokazatelja statusa željeza (serumsko željezo, TIBC, saturacija transferina, feritin); pokazatelja upale (broj leukocita i neutrofilnih granulocita, IL-6 i CRP-a), eritropoetske aktivnosti (broj retikulocita, topivi transferinski receptor i eritropoetin) i koncentracija hemoglobina. Svim bolesnicima s KOPB-om određeni su parcijalni tlak kisika i saturacija hemoglobina kisikom kao pokazatelji hipoksije.
Rezultati: Serumska koncentracija hepcidina bila je povišena u egzacerbaciji i stabilnoj fazi KOPB-a u odnosu na kontrolnu skupinu, te je pozitivno korelirala s IL-6 i CRP-om. Koncentracija hepcidina je bila pozitivno povezana s feritinom i negativno s TIBC-om. Eritropoetska aktivnost, mjerena apsolutnim brojem retikulocita bila je u svim fazama istraživanja niža od kontrolne skupine, a negativna povezanost je pokazana u egzacerbaciji. Koncentracija hepcidina nije bila povezana s parametrima hipoksije. U kontrolnoj skupini hepcidin je korelirao samo s pokazateljima statusa željeza, negativno s TIBC-om i pozitivno s feritinom.
Zaključak: Istraživanje je pokazalo da prisutna sistemska upala i povišena razina IL-6 u egzacerbaciji i stabilnoj fazi KOPB-a mogu biti odgovorne za opaženi porast koncentracije hepcidina. Porast koncentracije hepcidina mogao bi biti povezan s restriktivnom eritropoezom koja se očituje sniženim brojem retikulocita u svim fazama istraživanja, kao i snižavanju koncentracije hemoglobina u stabilnoj fazi bolesti. Dobiveni rezultati pružaju uvid u dinamičke promjene metabolizma željeza i povezanosti s razinom hepcidina. Sistemska upala prisutna kod velikog broja bolesnika s KOPB-om jača kako bolest napreduje, te bi porast razine IL-6 mogao voditi do daljnjeg porasta koncentracije hepcidina.
Background: Hepcidin is the main regulator of systemic iron homeostasis, and its expression is modulated by iron status, hypoxia, erythroid factors and inflammation. The aim of this study was to examine a relationship between level of hepcidin and iron status, erythropoietic activity, hypoxia and inflammation in exacerbations and stable COPD. We hypothesized that hepcidin concentration is changed compared to control group and is changing in acute exacerbation compared to stable COPD; hepcidin concentration is substantially influenced by inflammation and/or hypoxia.
Methods: The study included 40 COPD patients and 30 healthy subjects. In COPD group parameters were longitudinally monitored at three time points: at exacerbation, on resolution and in stable disease. We determined concentration of hepcidin and hemoglobin; parameters of iron status: serum iron, total iron binding capacity (TIBC), ferritin and calculated transferrin saturation. Soluble transferrin receptors, reticulocyte number (Rtc), and regulatory hormone erythropoietin were measured as indicators of erythropoietic activity. Systemic inflammation was assessed by determination of CRP, IL-6, and number of white blood cells and neutrophils. In COPD group partial oxygen pressure and haemoglobin oxygen saturation were determined.
Results: Hepcidin was elevated in exacerbations and in a stable phase compared to the control group and we found positive correlations of hepcidin with inflammatory markers IL-6 and CRP. Hepcidin also correlated positively with ferritin and inversely with TIBC. Erythropoietic activity, measured by absolute Rtc number, was significantly reduced in COPD compared to the control group in all study phases, and negative correlation with hepcidin was established in exacerbation. In exacerbation and stable disease hepcidin correlated with ferritin and TIBC. No correlations were observed with indices of hypoxia. In the control group, positive associations were observed only with indices of iron status, positive with ferritin and negative one with TIBC.
Conclusion: This study shows that elevated values of IL-6 present in exacerbations and stabile COPD might be responsible for the observed increased hepcidin level. These might be associated with restricted erythropoiesis as shown by lower number of reticulocytes and decreased level of haemoglobin at the stable phase. The results obtained might provide new insights into dynamic changes of iron metabolism and its relation to hepcidin level in COPD. Systemic inflammation present in majority of COPD patients increases over time as disease progresses so raising of IL-6 level could lead to further up-regulation of hepcidin.
Trenutno ne postoje farmakopejske ili standardne metode i aparature za ispitivanje oslobađanja djelatne tvari iz topikalnih mikročestica. Cilj ovog rada bio je razviti i validirati diskriminatornu in ...vitro metodu za ispitivanje oslobađanja djelatne tvari iz topikalnih mikročestica, uz korištenje imerzijske ćelije. Za potrebe rada pripremljene su i karakterizirane kitozanske i metakrilatne mikročestice s mupirocinom. Za izradu mikročestica korištena je tehnika sušenja raspršivanjem. Ispitana su sljedeća svojstva mikročestica: učinkovitost uklapanja lijeka, morfologija i veličina čestica, kristalno stanje, termička svojstva, spektralna svojstva, zeta potencijal, higroskopnost te sadržaj vode i ostatnih organskih otapala.
Kitozanske i metakrilatne mikročestice s mupirocinom korištene su kao modelni topikalni terapijski sustavi za razvoj in vitro metode za ispitivanje oslobađanja djelatne tvari. Imerzijska ćelija korištena je u kombinaciji s aparaturom s lopaticom. pH i temperatura medija (pH 5,5, 32°C) odabrani su u skladu s fiziološkim uvjetima na koži. Difuzija lijeka odvijala se preko membrane izrađene od smjese celuloznih estera, koja je pokazala nisku adsorpciju lijeka te nizak otpor difuziji lijeka. Nakon
početnog zastojnog vremena količina oslobođenog lijeka postala je proporcionalna korijenu iz vremena. Nagib u linearnom području krivulje oslobađanja lijeka korišten je kao mjera brzine oslobađanja. Varijacije u brzini okretanja lopatica (25 o/min, 50 o/min, 100 o/min), visini lopatica (1 cm, 2,5 cm) i volumenu medija za ispitivanje oslobađanja (100 ml, 200 ml) nisu značajno utjecale na brzinu oslobađanja. Analiza kitozanskih mikročestica izrađenih s kitozanima različitih molekulskih masa pokazala
je da se kod povećane molekulske mase kitozana smanjuje brzina oslobađanja. S druge strane, brzina oslobađanja bila je veća pri većoj koncentraciji lijeka unutar donorskog odjeljka. Na taj je način pokazana diskriminatornost metode prema razlikama u formulaciji, kao i prema razlikama u koncentraciji uzorka unutar donorskog odjeljka ćelije. Metodom je nadalje potvrđena sličnost serija istog sastava proizvedenih istim procesom. Metoda je validirana u skladu s ICH smjernicama. U sklopu validacije metode potvrđena je njena specifičnost, linearnost, točnost, preciznost i robusnost. Metoda je uspješno primijenjena kod kitozanskih i metakrilatnih čestica, čime je pokazan njen potencijal za karakterizaciju raznih tipova
topikalnih čestičnih terapijskih sustava. Razvijena metoda može biti koristan alat u razvoju formulacije kod takvih terapijskih sustava.
Currently there are no compendial or standard methods and apparatuses for in vitro release testing of topical microparticles. The aim of this study was to develop and validate a discriminative in vitro release method for topical microparticles using the immersion cell. For the purpose of this study chitosan-based and methacrylate-based microparticles with mupirocin were prepared by spray drying. The following characteristics of the microparticles were
examined: encapsulation efficiency, particle size and morphology, crystallinity, thermal properties, spectral properties, surface charge, hygroscopicity, residual organic solvents and water content. Chitosan-based and methacrylate-based microparticles with mupirocin were used as model topical delivery systems for in vitro release method development. The immersion cells were used in combination with paddle dissolution apparatus. The pH and temperature of the release medium (pH 5.5, 32°C) were selected to reflect the physiological skin conditions. Diffusion of the drug occured
across a mixed cellulose ester membrane, which demonstrated low drug adsorption and low diffusional resistance. After an initial lag phase the amount of drug released became proportional to the square root of time. The slope in the linear portion of the release curve was used as a measure of release rate. Variations in paddle rotation speed (25 rpm, 50 rpm, 100 rpm), paddle height (1 cm, 2.5 cm) and volume of release medium (100 ml, 200 ml) did not significantly alter the release rates. Appropriate discriminatory power of the method was confirmed as the method was able to detect differences in formulation, as well as differences in drug concentration inside the sample compartment. The analysis of chitosan-based microparticles prepared with chitosans of different molecular weights has shown that the release rate decreases with increasing molecular weight of chitosan. On the other hand, the release rate increased with increasing drug concentration inside the sample compartment. The method was further used to confirm sameness between batches of the same composition prepared by the same process. The method was validated for its specificity, linearity, accuracy, precision and robustness in line with International Conference on Harmonisation (ICH) guidelines. The method was successfully applied both for chitosan-based and methacrylate-based microparticles, which demonstrates its potential application for various types of topical particulate delivery system.
Novi dokazi ukazuju da je antinociceptivno djelovanje botulinum toksina tipa A (BT-A) središnjeg porijekla. U ovom doktorskom radu smo provjerili ovu pretpostavku u ranije nedovoljno istraženim ...oblicima boli, istraživali interakciju sa središnjim neurotransmitorima kao mogući mehanizam djelovanja te pokušali pobliže utvrditi mjesto djelovanja toksina u središnjem živčanom sustavu (SŽS).
Ispitivanja su izvedena na mužjacima Wistar štakora. U upalnoj, neuropatskoj i bilateralnoj mišićnoj boli ispitan je učinak selektivnih i neselektivnih antagonista opioidnih i GABAA receptora, primijenjenih sistemski, spinalno ili supraspinalno, na antinociceptivno djelovanje periferno (supkutano u šapu) primijenjenog BT-A. U tkivu kralješnične moždine ispitana je aktivacija neuronalnih i glija stanica, ekspresija mRNA proupalnih citokina i μ-opioidnih receptora te ekspresija Leu/Met-enkefalina metodama imunofluorescencije i lančane reakcije polimerazom s reverznom transkripcijom. Istraživano je antinociceptivno djelovanje BT-A nakon periferne, spinalne i supraspinalne primjene te je imunofluorescencijom ispitana enzimska aktivnost BT-A u tkivu SŽS-a.
Opioidni i GABAA antagonisti su ovisno o dozi, sistemski i intratekalno, ali ne i supraspinalno, poništili antinociceptivno djelovanje BT-A u svim ispitanim modelima. Učinak antagonista bio je kratkotrajan. BT-A je smanjio neuronalnu aktivaciju u dorzalnom rogu kralješnične moždine, što su antagonisti blokirali. BT-A je smanjio bol u dosad neistraženim modelima visceralne boli (peritonitis, kolitis). Bilateralno antinociceptivno djelovanje BT-A posljedica je prisutnosti toksina samo na ipsilateralnoj strani. BT-A je smanjio bol nakon periferne i intratekalne primjene, dok primijenjen supraspinalno (cisterna magna, moždane komore) nije djelovao, unatoč nalazu njegove enzimske aktivnosti u pojedinim regijama mozga uključenima u nocicepciju.
BT-A ima segmentalno antinociceptivno djelovanje spinalnoj razini, uz neizravnu aktivaciju endogenog opioidnog i GABA-ergičkog sustava. Ovi bi nalazi mogli biti važni za klinička ispitivanja potencijalno korisnih sinergističkih interakcija s konvencionalnim analgeticima i drugim lijekovima te usmjeriti klinička ispitivanja na nove indikacije i nove načine primjene, poput intratekalne.
Novel evidence suggests that the antinociceptive effect of botulinum toxin type A (BT-A) is of central origin. In this doctoral thesis, we verified this assumption in previously insufficiently investigated types of pain, investigated the interaction with central neurotransmitters as the possible mechanism of action and tried to determine the site of the toxin’s action within the central nervous system (CNS).
Male Wistar rats were used in experiments. We examined the effect of selective and nonselective opioid and GABAA antagonists, applied systemically, spinally and supraspinally, on antinociceptive effect of peripherally (subcutaneously into hind paw) applied BT-A in inflammatory, neuropathic, and bilateral pain. Neuronal and glial cells’ activation, proinflammatory cytokines’ and μ-opioid receptors mRNA expression, and Leu/Met-enkephalin protein expression were analyzed using immunofluorescence and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) in lumbar spinal cord tissue. Further, we investigated the antinociceptive effect of BT-A following peripheral, spinal and supraspinal application. In parallel, enzymatic activity of BT-A in CNS tissue was examined using immunofluorescence.
Opioid and GABAA antagonists, applied systemically and intrathecally, dose-dependently abolished the antinociceptive effect of peripheral BT-A in all tested models, while no effect was observed following their supraspinal application. The effect of antagonists was short-lasting. BT-A reduced neuronal activation in dorsal horn, which was abolished by both, opioid and GABAA antagonist. BT-A diminished pain in, yet uninvestigated, models of visceral pain (peritonitis and colitis). BT-A’s bilateral antinociceptive action occurs after toxin’s presence on ipsilateral side only. BT-A reduced pain after peripheral and intrathecal application, but not after application in cisterna magna or cerebral ventricles, despite of its enzymatic activity in brain regions involved in nociception.
BT-A has segmental antinociceptive effect at the spinal level, which involves an indirect activation of endogenous opioid and GABA-ergic systems. These findings might guide clinical investigations of potentially useful additive or synergistic effects with conventional analgesics and direct clinical trials for novel indications and the routes of application, like intrathecal.
