Masarykova univerzita Lékařská fakulta Úloha oxidačného stresu v patogenéze septických stavov, respiračného zlyhania na podklade vírusovej infekcie a bolestivých syndrómov Habilitačná práca podľa § 72 odst. 3 písm. b) zákona o vysokých školách 2024 MUDr. Ladislav Kočan, PhD. Poďakovanie Na tomto mieste by som sa rád poďakoval všetkým, ktorí ma podporovali a vytvorili vhodné podmienky pre realizáciu tejto práce. Ďakujem mojím školiteľom doc. MUDr. Jozefovi Firmentovi, PhD. prednostovi I. kliniky anestéziológie a intenzívnej medicíny Univerzitnej nemocnice Louisa Pasteura v Košiciach, MUDr. Róbertovi Rapčanovi PhD., FIPP medicínskemu riaditeľovi EuroPainClinics, doc. MUDr. Igorovi Martuliakovi PhD. prednostovi Kliniky Algeziológie Fakultnej nemocnice s poliklinikou F.D. Roosevelta v Banskej Bystrici, doc. MUDr. Pavlovi Tôrôkovi CSc. emeritnému prednostovi Kliniky anestéziológie a intenzívnej medicíny VUSCH a.s. z môjho domovského pracoviska, za ich podporu a odborné vedenie v mojej klinickej praxi, ako aj za ich cenné rady a pripomienky pri vypracovaní tejto práce. Zvlášť ďakujem Prof. RNDr. Janke Vaškovej, PhD. a doc. MVDr. Ladislavovi Vaškovi CSc. z Ústavu lekárskej a klinickej biochémie LF UPJŠ za ich dlhoročné priateľstvo a úzku spoluprácu a konzultácie pri písaní habilitačnej práce. Moja vďaka patrí aj mojím kolegom z pracovísk z KAJJVI VUSCH a.s. a EuroPainClinics, ktorí mi počas mojej klinickej praxe vytvárali príjemné pracovné prostredie. V neposlednom rade by som sa rád poďakoval svojej rodine, rodičom a manželke Hanke za ich lásku, pochopenie a podporu. 2 OBSAH 1 ÚVOD...........................................................................................................................5 2.1 Patofyziológia oxidačného stresu...............................................................................8 2.2 Kriticky chorý pacient................................................................................................9 2.3 Terapeutické využitie selénu.....................................................................................10 2.4 Klinické štúdie sledujúce efektivitu selénu..............................................................11 2.5 Štúdia SE-AOX.........................................................................................................13 2.5.1 Dizajn klinickej štúdie Se-AOX............................................................................13 2.5.2 Kazuistiky v súvislosti so štúdiou SE-AOX..........................................................14 2.5.3 Závery štúdie SE-AOX..........................................................................................32 2.5.4 Citácie štúdie SE-AOX v Metaanalýzach a Systémových prehľadoch.................50 3 RESPIRAČNÉ ZLYHANIE NA PODKLADE VÍRUSOVEJ INFEKCIE A JEHO LIEČBA..........................................................................................................................51 3.1 Viachladinová ventilácia pľúc..................................................................................51 3.2 COVID- 19..............................................................................................................53 3.3 Práce súvisiace s grantovými projektami..................................................................54 3.4 Oxidačný stres pri vírusovej pneumónii...................................................................55 3.5 Postpneumonická fibróza pľúc.................................................................................56 4 ÚLOHA OXIDAČNÉHO STRESU V CHRONICKEJ BOLESTI............................64 5 OXIDAČNÝ STRES A OPIOID Y.............................................................................90 5.1 Štúdia Opioid-Redox Study.....................................................................................95 6 ĎALŠIE PRÁCE SPOJENÉ S TEMATIKOU AKÚTNEJ A CHRONICKEJ BOLESTI......................................................................................................................105 6.1 Problematika bolesti...............................................................................................105 6.2 Projekty manažmentu terapie bolesti v nemocničnom zariadení VUSCH a.s........107 6.3 Multicentrické projekty spojené s liečbou a výskumom akútnej a chronickej bolesti ......................................................................................................................................121 7 UČEBNICOVÉ VYSOKOŠKOLSKÉ TEXTY.......................................................156 8 VZDELÁVACIE AKTIVITY TEMATICKY SA VZŤAHUJÚCE K HABILITAČNEJ PRÁCI A GRANTOVÉ PROJEKTY.............................................160 8.1 Prednášky na medzinárodných podujatiach............................................................160 8.2 Popularizačné aktivity tematicky sa vzťahujúce k habilitačnej práci.....................161 8.3 Grantové projekty...................................................................................................162 -> j) ZOZNAM POUŽITÝCH SKRATIEK A SYMBOLOV..............................................163 ZOZNAM OBRÁZKOV..............................................................................................164 ZOZNAM TABULIEK................................................................................................164 ZOZNAM PRÍLOH......................................................................................................164 Literatúra.......................................................................................................................165 Komentár......................................................................................................................172 Habilitation Thesis Abstract.........................................................................................174 4 1 ÚVOD Predkladaná habilitačná práca podľa § 72 ods. 3 písmena b) zákona o vysokých školách je súhrnom štúdií a odborných textov venujúcich sa aktuálnym témam v oblasti anestéziológie, intenzívnej medicíny a algeziológie. Pre rozsiahlosť súboru publikácií a ich tém je práca rozdelená do 5 celkov, ktoré korešpondujú s hlavnými cieľmi mojej klinicko-výskumnej činnosti. Reaktívne formy kyslíka a dusíka sú atómy, molekuly alebo ich fragmenty, ktoré majú jeden alebo viac nespárených elektrónov a sú schopné, hoci aj krátkej samostatnej existencie. Častokrát zohrávajú dôležitú úlohu pri rôznych fyziologických dejoch, akými sú procesy súvisiace s fagocytózou, podieľajú sa na širokom spektre signalizačných funkcií a mnohých mechanizmov, ktoré variabilným pôsobením v živých sústavách udržiavajú stav homeostázy. Na druhej strane nežiadúci efekt nadprodukcie týchto reaktívnych foriem, patrí medzi široko skúmané procesy v súvislosti s ich podielom na patogenetickom rozvoji rozličných ochorení. Dôsledok ich negatívneho pôsobenia sa označuje ako oxidačný stres, ktorý je definovaný ako nerovnováha medzi tvorbou a odstraňovaním voľných radikálov a ďalších reaktívnych látok. Oxidačný stres stojí na pozadí mnohých ťažkých chorobných stavov a zohráva dôležitú úlohu pri amplifikácii imunitných dejov. U kriticky chorých je známa jeho aktivačná úloha v patogenéze syndrómov SIRS, sepsa, septický šok a širokého spektra patologických stavov, akými sú akútna pankreatitída, neurozápalové procesy, infekčné pneumónie a ďalšie závažné diagnózy. Liečba týchto ochorení je veľmi komplexná a prístup je od pacienta k pacientovi individuálny. Svetovou zdravotníckou iniciatívou bola v rokoch 2002 - 2003 snaha o zvýšenie prežívania pacientov so sepsou. Táto iniciatíva viedla k vypracovaniu a prijatiu spoločného programu za prežitie sepsy pod názvom „Surviving Sepsis Campaign, ktorý pozostával z komplexných a jednotných odporúčaných postupov v diagnostike a liečbe ťažkej sepsy. Odporúčania boli prvýkrát publikované v roku 2004 a následne revidované podľa nových dôkazov Evidence Based Medicíne (EBM -medicíny založenej na dôkazoch) v rokoch 2008, 2012, 2017 a 2021 (1, 2). V priebehu týchto časových období boli přehodnocované rôzne postupy, vrátane antioxidačnej suplementačnej terapie formou enterálnej a parenterálnej aplikácie vitamínov, stopových prvkov (zinok a selén) a podávania glutamínu. Medzi dôležité 5 štúdie, zaoberajúcimi sa touto problematikou v kľúčovom období kritického hodnotenia dôkazov, sa radia štúdie autorov Valenta a kol. a Andrews a kol. (3, 4). Jednou z dôležitých prác nášho lekársko-výskumného kolektívu, ktoré podporili negatívne závery liečby selénom, patrí prospektívna observačná klinická štúdia Se-AOX, ktorej výsledky nepotvrdili významný benefit po suplementácii selénu vzhľadom na sledovanú mortalitu pacientov s ťažkou sepsou a septickým šokom. V období 2019 - 2022 svetová verejnosť čelila vírusovej pandemii COVID-19. V tomto období náš lekársko-výskumný tím pracoval na zlepšení ventilačných možností u pacientov s nehomogénnym poškodením pľúc v dvoch významných projektoch realizovaných v rámci „Operačného programu Integrovaná infrastruktura", ktoré boli spolufinancované zo zdrojov Európskeho fondu regionálneho rozvoja v spolupráci s Ministerstvom školstva, výskumu, vývoja a mládeže Slovenskej republiky. Molekulárne princípy prestavby pľúcneho tkaniva na menejcenné fibrózne tkanivo úzko súvisia s excesívnou zápalovou reakciou a rozvojom oxidačného stresu. V rámci prognózy a terapeutickej stratégie u pacientov s ťažkou vírusovou pneumóniou sme mohli v tomto období využiť nadobudnuté poznatky aj zo štúdie Se-AOX. Zo získaných skúseností počas pandemie H1N1 v roku 2009, ako aj skúseností s ventiláciou u septických pacientov s ARDS, bolo možné ďalej rozvíjať metódy viac-hladinovej umelej pľúcnej ventilácie. Jej včasné nasadenie zlepšovalo prežívanie pacientov a poukazovalo na nižšiu úroveň postpneumonickej fibrotickej prestavby pľúcneho tkaniva. Pôsobenie oxidačného stresuje nešpecifické s potenciálom zasiahnuť rôzne tkanivá a orgánové systémy. Z doterajších výskumov vyplýva, že oxidačný stres zohráva významnú úlohu aj pri chronifikácii bolesti. Sprievodné kaskádové reakcie sprevádzajúce oxidačný stres vyvolávajú negatívne zmeny v centrálnom nervovom systéme na neurónoch ako aj na bunkách glie, vzhľadom na ich vysokú citlivosť voči oxidačným vplyvom a metabolickým zmenám, ktoré prebiehajú v rámci neurozápalu. Táto téma bola rovnako predmetom záujmu našej výskumnej práce. Vplyv oxidačného stresu a možnosti jeho supresie pri rozvoji neurozápalu, neuropatickej bolesti a pooperačnej kognitívnej dysfunkcie u pacientov po kardiochirurgických a nekardiochirurgických výkonoch aktuálne skúmame s naším vedecko-lekárskym tímom v prebiehajúcej prospektívnej observačnej klinickej štúdii NeuOX-postSurg Trial. Z experimentálnych výskumov vyplýva, že oxidačný stres hrá dôležitú úlohu v rámci viacerých mechanizmov, ktoré sú zapojené do nociceptívnej modulácie a centrálnej senzitizácie. Zároveň pretrvávanie prooxidačného stavu umocňuje terapeutické používanie opiátov. Doterajšie dôkazy klinických a experimentálnych štúdií poukazujú na zvýšenú tvorbu reaktívnych foriem kyslíka a dusíka pri metabolizácii morfínu, ktoré sú výsledkom 6 katalyzovaných reakcií aktivovanými enzýmami NOS (syntázou oxidu dusnatého) a fosfolipázou D2. Predpokladá sa podobný prooxidačný efekt aj pri metabolizácii ďalších silný opiátov z iných farmakologických skupín. Tieto teórie overuje náš lekársko-vedecký tím v prospektívnej observačnej klinickej štúdii Opioid-Redox Study. Dôležitou súčasťou lekárskej a vedeckej práce, ktorá nadväzuj e na náš doteraj ší výskum je štúdium akútnej a chronickej bolesti v nemocničnom a ambulantnom prostredí. Medzi dôležité klinické štúdie o ktoré sa opiera náš ďalší výskum patrí štúdium pooperačnej bolesti sledovaný v klinickej štúdii VUSCH/POPT1 study, ktorý porovnáva skupiny kardiochirurgických pacientov po sternotómiach a torakotómiach, ktorí sú na konvenčnej analgetickej farmakoterapii a skupiny pacientov po regionálnych nervových blokádach a kryoablačných výkonoch zameraných na interkostálne nervy. Sledovanie klinického stavu a hodnotenie zmien v kvalite života u pacientov s chronickou bolesťou v ambulantnom prostredí sme realizovali vo viacerých klinických štúdiách. Tieto klinické štúdie boli zamerané prevažne na pacientov s bolesťou dolnej časti chrbta tzv. „low back pain" a pacientov s kritickou končatinovou ischémiou. Klinický stav pacientov s postlaminektomickým syndrómom hodnotila po epiduroskopickom terapeutickom výkone multicentrická randomizovaná dvojito zaslepená klinická štúdia EPCS II. Klinická štúdia sledovala efekt samotnej mechanickej lýzy verzus mechanickej lýzy vrátane aplikácie liečiv do epidurálneho priestoru. Terapiu lumbálneho fazetového syndrómu hodnotí aktuálne prebiehajúca prospektívna multicentrická randomizovaná klinická štúdia EPCS XI porovnávajúca 3 skupiny pacientov, ktorí podstupujú rádiofrekvenčnú abláciu, kryoabláciu mediálnych nervových ramienok dorzálnej vetvy spinálneho nervu a endoskopický debridement puzdra fazetového klbu. Ďalšou ukončenou štúdiou zameranou na chirurgickú liečbu akútneho radikulárneho syndrómu bola prospektívna observačná štúdia EPCS V, ktorá hodnotila pooperačný stav pacientov a komplikácie po endoskopickej diskektómii. V týchto štúdiách bola hodnotená kvalita života prostredníctvom dotazníka EQ-5D-5L výskumnej skupiny EuroQuol, pričom v spolupráci so Slovenskou Akadémiou Vied boli stanovené koeficienty štatistických váh pre región Slovenskej a Českej republiky. Aktuálne realizujeme prospektívnu observačnú klinickú štúdiu: Tevi-LuSy-Study v spolupráci Východoslovenským ústavom srdcových a cievnych chorôb a s Technickou Univerzitou v Košiciach, ktorá sa zoberá klinickým hodnotením stavu pacientov a hodnotením úrovne perfúzie dolných končatín prostredníctvom termálnych zmien u pacientov s kritickou ischémiou dolných končatín po sympatikolytických intervenciách. Tento projekt je finančne podporený grantom Slovenskej spoločnosti pre štúdium a liečbu bolesti. 7 2 OXIDAČNÝ STRES V PATOGENÉZE SEPTICKÝCH STAVOV 2.1 Patofyziológia oxidačného stresu Reaktívne formy odvodené od kyslíka (ROS, reactive oxygen species) vznikajú za aeróbnych podmienok v regulovaných aj neregulovaných procesoch. Medzi ROS patria atómy alebo molekuly obsahujúce jeden alebo viac nespárených elektrónov. Sú produktami látkového metabolizmus a v živých organizmoch participujú na širokom spektre biologických procesov. Patria medzi ne radikálové formy ako superoxidový radikál (O2""), hydroxylový radikál (HO'), oxid dusnatý (NO) alebo neradikálové formy, napr. peroxid vodíka (H2O2) (5). ROS zohrávajú dôležitú úlohu v patogenéze širokého spektra chorobných stavov, vrátane závažných ochorení u kriticky chorých pacientov. Charakteristická je pre nich vysoká reaktivita a veľmi krátky polčas rozpadu. Medzi ich najvýznamnejšie funkcie patria antimikrobiálna ochrana a signalizačné funkcie (6). Tvorba ROS prebieha prevažne štyrmi hlavnými cestami, a to: V dýchacom reťazci prebiehajúcom v mitochondriách každej bunky, kde O2"" vzniká ako vedľajší produkt reakcie molekulárneho kyslíka a semichinónu. Významná je ale aj produkcia O2'" v polymorfonukleárnych leukocytoch, kde majú antimikrobiálnu funkciu. Druhý spôsob je zabezpečený prostredníctvom, reakcií katalyzovaných enzýmami rodiny NOX, NADPH-oxidázami, ktorými disponujú najmä makrofágy aneutrofilné leukocyty. Touto reakciou vzniká excesívne množstvo O2'", ktorý zohráva dôležitú baktericídnu úlohu. Tretia cesta tvorby ROS je katalyzovaná enzýmom xantínoxidázou, ktorá je aktivovaná napríklad počas ischemických podmienok spojených s následnou reperfúziou tkanív. Rovnako aj pri tejto reakcii vzniká masívne množstvo O2"" v čase reperfúzie. Jej najväčšia aktivita sa predpokladá najmä pri polytraume a pri kardiochirurgických výkonoch. Štvrtou variantou je tvorba ROS pri poškodení buniek. Procesy sú spojené s uvoľňovaním intracelulárnych redoxne aktívnych iónov (Fe, Cu), ktoré amplifikujú tvorbu voľných radikálov v Haber-Weissovej a Fentonovej reakcii, kedy dochádza ku konverzii peroxidu vodíka na hydroxylový radikál (7). ROS majú schopnosť poškodzovať proteiny, polysacharidy, nukleové kyseliny, nenasýtené mastné kyseliny, čo vedie k dramatickému poškodeniu buniek. Navyše ROS môžu iniciovať uvoľňovanie cytokínov z imunokompetentných buniek, aktivovať zápalovú kaskádu a 8 zvyšovať expresiu adhéznych molekúl na povrchu buniek. Voľné radikály spúšťajú kaskádu intercelulárnych udalostí, spôsobujúcich uvoľnenie nukleárneho transkripčného faktora NF-kB z jeho inhibičného proteinu IkB. NF-kB faktor sa translokuje do bunkového jadra a naviaže sa na DNA, čo spúšťa iniciáciu transkripcie génov, ktoré sú dôležité z hľadiska zápalu. NF-kB kontroluje produkciu mediátorov akútnej fázy ako TNF-a, IL-2 a IL-2 receptorov, ktoré opäť aktivujú NF-kB, čím amplifikujú zápalovú kaskádu . Poškodenie tkanív a zápal vedú ku akumulácii granulocytov v danom tkanive, čo vedie k zvýšenej produkcii ROS a bludnému kruhu ďalšieho oxidačného poškodenia tkaniva (7, 8). 2.2 Kriticky chorý pacient Kritické ochorenia sa vyznačujú komplexom porúch vrátane excesívnej zápalovej odpovede, dysfunkcie mitochondrií, bunkovej imunity a rozvojom oxidačného stresu. Oxidačný stres nemusí byť len dôsledkom, ale čoraz viac sa uznáva ako kľúčový prispievateľ iniciačných patofyziologických procesov, ktoré sú základom vzniku kritického ochorenia (9) (10). Príkladom sledu týchto kaskádových reakcií je syndróm systémovej zápalovej odpovede (SIRS). Z klinického hľadiska je medicínsky manažment kriticky chorých pacientov na intenzivistickom lôžku zameraný na liečbu širokého spektra závažných klinických stavov, ktoré ohrozujú jedinca na živote, a to zlyhávaním jedného alebo viacerých orgánových systémov s možnými prejavmi kardiogénneho, hypovolemického, distribučného, obštrukčného šoku alebo ich kombinácie (11). Medzi najčastejšie príčiny hospitalizácie na oddeleniach intenzívnej medicíny patria stavy spojené s respiračným a kardiálnym zlyhaním. Ďalšími závažnými ochoreniami vyžadujúcimi intenzívnu terapiu sú septické stavy, ťažké traumy, maligně poruchy srdcového rytmu, poranenia CNS, cievne mozgové príhody, rozsiahle nekardiochirurgické a kardiochirurgické operačné výkony, popáleniny a ďalšie stavy vyžadujúce rozsiahly monitoring vitálnych funkcií, ventilačnú podporu, farmakologickú vazopresorickú a inotropnú podporu, mechanické podpory obehu a srdca, mimotelovú eliminačnú liečbu, vrátane komplexnej nutričnej podpory. Pacienti sú vystavení noxam, pri ktorých dochádza k nadmernej tvorbe voľných radikálov, zároveň sú ohrození malnutríciou, deficitom mikronutrientov a vyčerpaním endogénnych zásob molekúl s antioxidačnými vlastnosťami (12, 13). V klinických štúdiách sledujúcich závažné klinické stavy bol hodnotený terapeutický efekt viacerých látok, ktoré disponujú antioxidačnými vlastnosťami. Medzi významné látky, ktoré 9 sú súčasťou antioxidačných systémov, patria vitamíny C, E, P-karotén, kyselina eikozapentaénová, zinok, selén a glutamín (5, 12 -14). 2.3 Terapeutické využitie selénu Organizmus disponuje súborom komplexných obranných mechanizmov, zahŕňajúcich intra- a extracelulárne zložky, ktoré dokážu za fyziologických podmienok efektívne eliminovať tvorbu reaktívnych foriem kyslíka a dusíka. Týmto spôsobom je ich celková kvantita v určitom priestore regulovaná, čo bráni rozvoju nekontrolovaných kaskádových oxidačných reakcií. Esenciálne stopové prvky ako meď, mangán, zinok, železo a selén sú kľúčové pre aktivitu enzýmov, ako je superoxiddismutáza (SOD), kataláza a glutatiónperoxidáza (GPx). Okrem toho neenzymatické obranné mechanizmy zahŕňajú molekuly ako glutatión, albumín, vitamíny E, C a P-karotén. Na pozadí SIRS môže nedostatok týchto endogénnych antioxidantov vzniknúť v dôsledku faktorov, ako je presakovanie kapilár, hemodilúcia, neadekvátny príjem stravy a lekárske zásahy, ako je kontinuálna renálna substitučná terapia. Najzávažnejšie prípady SIRS sa často zhodujú s najvýraznejším úbytkom antioxidantov (9, 15). Medzi ďalšie kritické stavy patrí sepsa a septický šok. Charakteristickým prejavom sepsy je život ohrozujúce zlyhávanie orgánov, ktoré je spôsobené neprimeranou odpoveďou hostiteľa na infekciu (16). Progresiou sepsy do septického šoku dochádza k poruchám krvného obehu a k bunkovým či metabolickým zmenám, ktoré zvyšujú riziko úmrtia. Sepsa napriek enormne vynaloženému úsiliu predstavuje závažný medicínsky a socioekonomický problém. Od roku 2002 bola formou Barcelonskej výzvy odštartovaná kampaň za prežitie sepsy „Surviving Sepsis Campaign" (SSC), zaoberajúca sa možnosťami liečby ťažkých septických stavov s cieľom zníženia mortality. Tento program zahŕňal vytváranie jednotných odporúčaní zameraných na včasnú diagnostiku a liečbu sepsy na podklade medicíny založenej na dôkazoch. Okrem včasnej identifikácie vyvolávajúcich agensov, empirickej a cielenej antimikrobiálnej liečby, včasnej tekutinovej resuscitácie obehu a udržania cieľových hodnôt stredného arteriálneho tlaku, boli posudzované ďalšie predpokladané terapeutické postupy a aplikácia liečiv, ktoré by mohli pozitívne ovplyvniť priebeh ochorenia (1). Medzi sledovanými látkami boli aj v tomto čase látky s antioxidačnými vlastnosťami ako vitamín C, E, alebo látky, ktoré k syntéze antioxidačne pôsobiacich molekúl sú potrebné, ako glutamín a mikroprvky zinok a selén (17). 10 Práve selén ako kofaktor významných anti oxidačný ch selenoenzýmov sa javil na základe výsledkov vtedajších veľkých klinických štúdií ako tzv "magic bullet" alebo "game changer", ktorý by mohol zvrátiť nepriaznivý priebeh septických ochorení. Avšak na vyslovenie jednoznačných účinkov chýbali ďalšie dôkazy. 2.4 Klinické štúdie sledujúce efektivitu selénu Terapeutické ovplyvnenie oxidačného stresu u kriticky chorých pacientov je dlhodobo predmetom klinického výskumu. Pozitívny terapeutický efekt suplementovaného selénu bol na prelome 20 a 21 storočia zaznamenaný v niekoľkých významných štúdiách. V roku 1994 boli publikované výsledky klinickej štúdie zaoberajúcej sa prospektívnym sledovaním pacientov s diagnostikovanou akútnou pankreatitídou. U pacientov bol počas liečby parenterálne suplementovaný pentahydrát seleničitanu sodného. Štúdia prebiehala na jednotkách intenzívnej starostlivosti v Nemeckých mestách v Rostocku a v Drážd'anoch. Celkovo bolo do štúdie zaradených 330 pacientov. Po stanovení diagnózy bol pacientom podaný bolusovou formou 200 ug pentahydrátu seleničitanu sodného a následne 800 ug kontinuálne počas nasledujúcich 24 hodín. Od druhého dňa sa podávalo 500 ug pentahydrátu seleničitanu sodného denne. S dobre načasovanou terapiou selénom miera mortality, komplikácií a počet následných operácií dramaticky poklesol. Mortalita v skupine 245 pacientov z Rostocku klesla na nulu. V Drážd'anoch po suplementácii selénu bolo v sledovanej skupine zaznamenaných 8 úmrtí z celkového súboru 85 pacientov, ktorí boli zaradení do štúdie. Komplikácie sa pridružili, až keď sa terapia začala príliš neskoro alebo ak sa jednalo o biliárne formy pankreatitídy (18). Ďalšie pozitívne výsledky parenterálnej suplementácie selénu publikoval Angstwurm v roku 2007 v časopise Critical Care zhrnutím záverov prospektívnej randomizovanej, placebom kontrolovanej multicentrickej štúdie. Celkovo bolo v tejto štúdii zaradených 249 pacientov so SIRS, sepsou a septickým šokom, ktorých APACHEIII bol viac ako 70 bodov. Pacientom bol po stanovení diagnózy podaný bolus 1000 ug pentahydrátu seleničitanu sodného počas 30 minút v kontinuálnej infúzii a nasledujúcich 14 dní denne bola aplikovaná kontinuálna infúzia 1000 ug pentahydrátu seleničitanu sodného. Počas štúdie neboli zistené nežiaduce účinky alebo známky predávkovania selénom. Po analýze podľa pôvodného liečebného zámeru bola stanovená mortalita v placebo skupine (50,0 %) versus skupina liečená selénom (39,7 %) (p = 0,109; odds ratio, 0,66; interval spoľahlivosti, 0,39-1,1). Ďalšie analýzy podskupín odhalili významné zníženie úmrtnosti medzi pacientmi so septickým 11 šokom komplikovaným diseminovanou intravaskulárnou koaguláciou (n = 82, p = 0,018), ako aj medzi najkritickejšie chorými pacientmi so skóre APACHE III > 102 (horný kvartil, n = 54, p = 0,040) alebo u pacientov, ktorí mali dysfunkciu viac ako troch orgánov (n = 83, p = 0,039) (19). Sakr v roku 2007 v British Journal of Anaesthesia publikoval výsledky prospektívnej observačnej štúdie, do ktorej bolo zaradených 60 pacientov rozdelených do skupín podľa závažnosti klinického stavu. Jednalo sa o pacientov s nekomplikovaným priebehom SIRS, ťažkým priebehom SIRS, ťažkou sepsou a septickým šokom. U všetkých týchto pacientov boli počas hospitalizácie dokumentované znížené hladiny plazmatického selénu oproti referenčnej hladine zdravých jedincov. Zistením bolo, že koncentrácia plazmatických koncentrácií selénu klesala v závislosti od dĺžky hospitalizácie, pričom výraznejší pokles bol pozorovaný u pacientov s orgánovým zlyhaním, najmä v dôsledku infekcií. Nižšie hladiny selénu v plazme boli spojené so zvýšeným poškodením tkaniva, prítomnosťou infekcie alebo orgánovej dysfunkcie/zlyhania a zvýšenou mortalitou na JIS (20). Optimizmus v odborných kruhoch začal upadať s pribúdaním ďalších štúdií, ktorých výsledky neboli natoľko presvedčivé a nepriniesli očakávaný benefit. Príkladom je multicentrická, dvojito zaslepená štúdia, s kontrolnou placebo skupinou, ktorá sa uskutočnila na 11 klinikách intenzívnej starostlivosti vo Francúzsku. Táto štúdia bola zameraná na podávanie selénu u pacientov v septickom šoku. V štúdii sa podávalo 4000 ug pentahydrátu seleničitanu sodného ako bolus v úvodnej dávke a 1000 [ig na deň v kontinuálnej infúzii počas 9 dní. V závere nebol zaznamenaný žiadny signifikantný rozdiel skrátenia času liečby medzi oboma skupinami po vysadení vazopresorov. Nebol zaznamenaný žiadny pozitívny efekt na dobu pripojenia pacienta na mechanickú ventiláciu, ani dĺžku hospitalizácie, pobytu na JIS, vplyvu na výskyt nozokomiálnych pneumónií, alebo zlepšenia renálnych funkcií u pacientov s renálnou insuficienciou. Nebola zistená toxicita, ale ani sa nepotvrdil pozitívny klinický výsledok u pacientov v septickom šoku (21). 12 2.5 Štúdia SE-AOX 2.5.1 Dizajn klinickej štúdie Se-AOX Vzhľadom na rozporuplné výsledky doterajších klinických štúdií zaoberajúcich sa suplementáciou selénu a jeho perspektívny terapeutický potenciál v teoretických modeloch, náš vedecko-lekársky tím pripravil dizajn prospektívnej observačnej klinickej štúdie zaoberajúcej sa hypotézou účinnosti liečby selénom u kriticky chorých pacientov s ťažkou sepsou alebo septickým šokom. V roku 2008 bola schválená Prospektívna observačná klinická štúdia: Therapeutic Effect of Sodium Selenite on Oxidative Stress in Patients With Severe Sepsis, Akronym: Se-AOX, ktorá bola registrovaná v databáze clinicaltrials.gov pod registračným číslom NCT02026856. Cieľom tejto štúdie bolo: porovnanie klinických výsledkov u dvoch skupín pacientov so sepsou, pričom prvej skupine bol suplementovaný pentahydrát seleničitanu sodného v kontinuálnej dávke 750 ug/24 hod počas 6 dní. Druhú skupinu tvorila placebo skupina pacientov. Primárne boli pacienti rozdelení do 4 podskupín, podľa toho, či podstúpili alebo nepodstúpili chirurgický výkon, selén versus placebo. sepsa / septický šok Obrázok č.l: Flow diagram štúdie SE-AOX - prospektívnej analýzy. Po zozbieraní a vyhodnotení údajov tejto časti štúdie, sme ďalej hodnotili efektivitu liečby vzhľadom na úroveň oxygenácie arteriálnej krvi v pľúcach. Pacienti boli sekundárne retrospektívne rozdelení do 4 podskupín podľa hodnôt oxygenačného indexu (pomeru parciálneho tlaku kyslíka v arteriálnej krvi a frakcie inspirovaného kyslíka) Skupina 01 < 200, OI > 200, selén versus placebo. 13 sepsa / septický šok Obrázok č.2: Flow diagram štúdie SE-AOX - retrospektívnej analýzy. V jednotlivých skupinách a podskupinách bola monitorovaná intenzita oxidačného stresu prostredníctvom aktivity antioxidačných enzýmov, a to GPx, glutatiónreduktázy (GR) a SOD v krvnej plazme. Hodnotené boli dynamické zmeny vybraných laboratórnych parametrov, a to C-reaktívneho proteinu, laktátu, urey, kreatininu, fibrinogénu, albumínu a celkových bielkovín, celkového počtu leukocytov a ich populácií, pomeru neutrofilov k lymfocytom a počtu trombocytov. Ďalším cieľom štúdie bolo hodnotenie klinických výsledkov v jednotlivých skupinách a podskupinách, a to výskyt infekčných komplikácií, respiračných a renálnych dysfunkcií a mortalita pacientov. 2.5.2 Kazuistiky v súvislosti so štúdiou SE-AOX Počas fázy klinickej štúdie, v ktorej sme realizovali zber údajov od pacientov, bolo zaznamenaných niekoľko pozoruhodných prípadov, v ktorých pozitívny zvrat kritického stavu nastal po aplikovaní komplexnej intenzivistickej liečby vrátane suplementácie pentahydrátu seleničitanu sodného a glutamínu. Prvá kazuistika popisuje úspešnú liečbu pacienta s ťažkou akútnou nekrotizujúcou pankreatitídou a s pridruženým mul ti orgánovým zlyhaním, kvalitatívnou poruchou vedomia, rozvinutým šokovým stavom, s úvodným APACHE skóre 19 bodov a SOFA skóre 6 bodov. V úvode liečby bol pacient napojený na umelú pľúcnu ventiláciu pre závažnú respiračnú insuficienciu (Pa02/Fi02 = 70) s klinickým a RTG obrazom ARDS. Pre potvrdenú difúznu peritonitídu bola pacientovi po prijatí vykonaná abdominálna laparotómia s následnou lavážou brušnej dutiny. Nasledujúca liečba ďalej zahŕňala podporu respiračných funkcií formou umelej pľúcnej ventilácie, kombinovanej cielenej antimikrobiálnej terapie podľa zistených aktuálnych kultivácii a ich citlivostí. Pre pridružené obličkové zlyhanie pacient bol dialyzovaný formou CVVHD. Pacientovi bola aplikovaná komplexná parenterálna výživa 14 spolu s adjuvantnou terapiou, ktorá zahŕňala suplementáciu selénu intravenóznou aplikáciou pentahydrátu seleničitanu sodného v dávke 750 ug/deň v kontinuálnej infúzii počas šiestich dní. Zároveň bol v infúznom roztoku parenterálne podávaný alanylglutamín v dennej dávke 100 ml počas šiestich dní v tom istom čase. Počas obdobia suplementácie bola monitorovaná dynamika zápalových buniek (leukocyty, pomer neutrofilov a lymfocytov, trombocyty) a zmena dynamiky biochemických markerov a antioxidačných enzýmov (prokalcitonín, fibrinogén, CRP, laktát, GPx, GR). Priame meranie dynamiky antioxidačných enzýmov u pacienta z kazuistiky poukázalo na ich aktiváciu suplementovanými mikronutrientmi. Zároveň bol tento jav sprevádzaný poklesom plazmatických hladín prokalcitonínu a CRP. Dynamické trendy zvýšenia aktivít GPx a GR od aplikácie prvej dávky selénu a glutamínu dokazovala zvýšenú aktivitu antioxidačného systému. (Aktivita GPx pred suplementáciou -0,55 ukat/1, 2. deň suplementácie - 2,66 ukat/1, 7. deň od začatia liečby - 3 ukat/1. Aktivita GR pred suplementáciou - 0,1813 ukat/1, 2. deň suplementácie - 0,3 ukat/1, 7. deň od začatia liečby -1,65 ukat/1.) Pomer neutrofilov a lymfocytov vykazoval priaznivú klesajúcu tendenciu aj napriek stúpajúcej hodnote leukocytov. Stanovenie tohto pomeru korelovalo s intenzitou závažnosti systémového zápalu. V tejto kazuistike bola pozorovaná súvislosť medzi závažnosťou klinického stavu a mierou neutrofílie a lymfocytopénie. Pacient bol po 10 dňoch extubovaný, s GCS 15b, cirkulačné stabilný, bez vazopresorickej podpory a s negatívnymi zápalovými parametrami bol preložený na oddelenie chirurgie. Druhá kazuistika popisuje prípad 66-ročnej ženy, u ktorej sa po kolonoskopickom vyšetrení s polypektómiou rozvinul septický šok s cirkulačnou nestabilitou a multiorgánovým zlyhaním. U pacientky bola realizovaná exploratívna abdominálna laparotómia, ďalšia intenzivistická liečba zahŕňala cielenú antibiotickú liečbu, protektívnu umelú pľúcnu ventiláciu, mimotelovú eliminačnú liečbu, komplexnú parenterálnu a enterálnu výživu, vrátane suplementácie selénu vo forme pentahydrátu seleničitanu sodného v dávke 750 ug/deň počas šiestich dní, čo zodpovedá 250 ug selénu na deň. Parenterálna výživa bola obohatená o glutamín a jeho prekurzory, a to podávaním alanylglutamínu v dennej dávke 2 g. Pacientkin stav sa postupne zlepšoval, vrátane hemodynamických parametrov, bola vysadená vazopresorická podpora, operatívne realizovaná deeskalácia antibiotickej liečby pri poklese zápalových parametrov CRP a PCT, počtu leukocytov, renálnych a hepatálnych parametrov. Pacientka bola postupne odpojená od umelej pľúcnej ventilácie. Šestnásty deň hospitalizácie bola pacientka v zlepšenom stave preložená do spádovej nemocnice. Tretia kazuistika popisuje prípad 31 - ročnej pacientky s exacerbovanou Stillovou chorobou na dlhodobej imunosupresívnej liečbe. Pacientka bola zaradená do štúdie po náhlom 15 rozvoji syndrómu systémovej zápalovej odpovede s multiorgánovým zlyhávaním s manifestným hepatorenálnym syndrómom, respiračným zlyhaním, diseminovanou intravaskulárnou koagulopatiou v obraze prebiehajúceho distribučného šoku. Pacientke bola poskytnutá komplexná intenzi vi stická liečba vrátane enterálnej a parenterálnej výživy so suplementáciou pentahydrátu seleničitanu sodného v dávke 750 ug/deň počas šiestich dní. Nutrične prípravky obsahovali vyšší podiel rozvetvených esenciálnych aminokyselín (valín, leucín, izoleucín) a prekurzorov glutatiónu (cystein, glutamín). Počas liečby sme zaznamenali zvýšenie aktivít antioxidačných enzýmov a to aktivity GPx z východiskovej hodnoty 0,117ukat/l na 0,27 ukat/1, aktivity GR z úvodnej 0,267ukat/l na 0,683 ukat/1 a aktivity SOD z 3,98 ukat/1 na 4,45 ukat/1. Tento pozitívny trend korešpondoval s postupne sa zlepšujúcim klinickým stavom až do úplného vysadenia vazopresorickej podpory, zlepšenia respiračných, hepatálnych a renálnych parametrov. 16 Suplementácia selenu u pacientov s ťažkou akútnou pankreatitídou Kočan L., Firment J., Simonová J., Vašková J., Guzy J. I. klinika anestéziológie a intenzívnej medicíny, Fakultná nemocnica L. Pasteura, Košice, Slovenská republika, prednosta kliniky: doc. MUDr. Jozef Firment, Ph.D. Súhrn Kočan L., Firment J., Simonová J., Vašková J., Guzy J.: Suplementácia selénu u pacientov s ťažkou akútnou pankreatitídou Suplementácia selénu zlepšuje antioxidačný status u kriticky chorých pacientov s ťažkou akútnou pankreatitídou v závislosti od dávky a doby podávania selénu. Cieľom práce je poukázať na prínos antioxidaČnej terapie hradením selénu. Metóda: Pacient s akútnou pankreatitídou a rozvíjajúcim sa septickým šokom bol prijatý na oddelenie intenzívnej medicíny. V rámci adjuvantnej terapie bola zahájená suplementácia selénu v kontinuálnej infúzii v dávke 750 @g/24 hodín počas šiestich dní. Meraním bolo zistené zvýšenie aktivity antioxidačného enzýmu glutatión peroxidázy a zníženie zápalových markerov v čase suplementácie. Práca poukazuje na možnosti ovplyvnenia patogenézy syndrómu systémovej zápalovej odpovede v jeho počiatočnej fáze a ovplyvnenie vývoja chorobného stavu u pacientov s ťažkou akútnou pankreatitídou. Kľúčové slová: ťažká akútna pankreatitída - sepsa - syndróm systémovej zápalovej odpovede - selén - glutatión peroxidáza Summary Kočan L., Firment J., Simonová J., Vašková J., Guzy J.: Selenium Supplementation in Patients with Severe Acute Pancreatitis Selenium supplementation improves antioxidant status in critically ill patients with severe acute pancreatitis. It depends on quantum of dosage and supplementation time. The aim of this analysis is point out on benefit of antioxidant therapy by supplementing selenium. Methods: Patient with severe acute pancreatitis and developing septic shock was admitted on anesthesiology and intensive care department. Adjuvant supplementation therapy with selenium was started in continual infusion 750 ug/24 h during next six days. Activity of antioxidant enzyme glutathione peroxidase and others inflammatory markers were decrease. A case report presents the possibility to affect on systemic inflammatory response syndrome pathogenesis in initial phase. It has to improve therapeutic progress in patients with severe acute pancreatitis. Key words: severe acute pancreatitis - sepsis - systemic inflammatory response syndrome - selenium - glutathione peroxidase Rozhl. Chir., 2010, roč. 89, č. 1, s. ÚVOD Ťažká akútna pankreatitída predstavuje závažný medicínsky problém z hľadiska vážnosti stavu, rapídnej progresie a vysokej mortality [1, 2, 3]. Stratégia liečby vyžaduje komplexný multidisplinárny prístup. Pacientov je nutné hospitalizovať na oddeleniach, ktoré túto problematiku zvládajú, a to chirurgického alebo interného typu. Najťažšie formy akútnej pankreatitídy spojené s respiračným zlyhaním a multiorgánovou dys-funkciou patria na lôžkové časti oddelení anestéziológie a intenzívnej medicíny [4]. Ochorenie sa spája s rozvojom syndrómu systémovej zápalovej odpovede (SIRS), v patogenéze ktorého dochádza k nadmernej aktivácii imunitných dejov, zápalových mediátorov a vzniku oxidačného stresu, ktorý sa v úvodnej fáze významne podieľa na amplifikácii zápalových dejov [5]. Vážnou komplikáciou ťažkej akútnej pankreatitídy je multiorgánové zlyhanie, ktoré vzniká na podklade SIRS a to vo včasnej fáze priebehu 518 ochorenia [6]. Antioxidačná terapia má potenciál zasiahnuť prostredníctvom vychytávania voľných radikálov do priebehu počiatočnej fázy rozvoja SIRS, čo môže výrazne ovplyvniť prežívanie pacientov [7]. Klinicky významný benefit bol dokázaný substitúciou troch živín s antioxidačnými vlastnosťami. Pri podávaní selénu sa zlepšujú klinické výsledky pri infekciách a orgánovom zlyhaní, pri podávaní glutamínu v rozsiahlych štúdiách bolo zistené zníženie infekčných komplikácií u kriticky chorých pacientov. V súvislosti s kyselinou eikosapentaenovou a mikronutrientmi bol zaznamenaný výrazný protizápalový efekt a otupená odozva endotoxínu [8]. Z podávaných mikronutiientov, sá zdá byť selén najviac účinnou antioxidačnou látkou v klinickom merítku, po ňom nasleduje zinok, vitamíny C a E a 6-karotén. Cieľom práce je poukázať prostreníctvom kazuistiky na prínos suplementačnej adjuvantnej terapie vhodnými mikronutrientmi u kriticky chorých pacientov [9]. Rozhledy v chirurgii 17 METODIKA Pacientovi bol počas adjuvantnej terapie v dennej dávke 750 @g v kontinuálnej infúzii počas šiestich dní suplementovaný selén vo forme pentahydrátu seleniči-tanu sodného. Zároveň bol v infúznom roztoku podávaný alanylglutamín do centrálneho venózneho katétra v dennej dávke 100 ml počas šiestich dní v tom istom čase. Počas obdobia suplementácie bola monitorovaná dynamika zápalových buniek (leukocyty, pomer neu-trofilov a lymfocytov, trombocyty) a zmena dynamiky biochemických markerov a antioxidačných enzýmov (prokalcitonín, fibrinogen, CRP, laktát, glutatiónpero-xidáza, glutatión reduktáza). KAZUISTIKA Pacient vo veku 50 rokov s akútnou hemoragicko-ne-krotizujúcou pankreatitídou a pridruženým multiorgá-novým zlyhávaním bol preložený na I. KAIM FNLP z OAIM NsP Michalovce, kde bol operovaný pre difúznu peritonitídu. Bola vykonaná drenáž brušnej dutiny s lavážou a zavedením preplachových drénov. Pri prijatí bol pacient v komatóznom stave, APACHE 19 bodov a SOFA skóre 6 bodov na agresívnej umelej pľúcnej ventilácii (UPV) s cirkuláciou podporovanou no-radrenalínom. Bolo realizované vstupné CT vyšetrenia abdomenu, pľúc a mozgu. Po desiatich dňoch hospitalizácie pre anemizáciu a pridružený septický šok chirurg indikuje revíznu operáciu. Bola realizovaná drenáž omentálnej burzy so zavedením preplachovej drenáže a súčasne bola vykonaná punkčná jejunostómia. Ihneď po operácii sa realizovaná adjuvantná antioxidačná terapia kontinuálnou su-plementáciou selénu v dávke 750 @g/24 hodín počas nasledujúcich šiestich dní. Pravidelne boli merané aktivity antioxidačných enzýmov ako aj štandardné zápalové parametre (Tab. 1). Počas hospitalizácie sa rozvíja obojstranný fluidothorax viac vľavo, preto boli realizované opakovane evakuačné punkcie aj v spoluprá- ci s hrudníkovým chirurgom. Bola aplikovaná kombinovaná ATB liečba a bolo pridávané antimykotikum (fluconazol) vzhľadom na kandidovú infekciu. Pre pridružené akútne obličkové poškodenie je pacient hemo-dialyzovaný, v úvode bola aplikovaná mimotelová eliminačná liečba (MEL) formou CVVHD a neskôr MEL formou intermitentnej hemodialýzy. Pacient sa v ďalšom priebehu cirkulačné stabilizuje a už nevyžaduje va-zopresorickú podporu. Po týždni opätovne dochádza k anemizácii pacienta, kontrolné CT vyšetrenie abdomenu odhalilo subkapsu-lárneho hematóm sleziny. Konziliárny chirurg odporučil konzervatívny postup. Vzhľadom na vzostup zápalových markerov je napokon pacient revidovaný, retro-peritoneálny absces bol vydrénovaný a bola ponechaná preplachová drenáž s Betadinovým roztokom. Na oddelení intenzívnej jednotky pokračujeme v ATB liečbe a podpornej UPV. Z hľadiska ďalšej prognózy dlhodobej a potreby podpornej UPV je vykonaná perkutánna dilatačná tracheostómia. Priebeh hospitalizácie bol komplikovaný hematemézou a melénou, pri endosko-pickom náleze bol zistený vred v bulbe duodéna a ero-zívna ezofagitída. Krvácanie z horného GIT-u bolo zvládnuté konzervatívne. Pri kontrolnom CT vyšetrení pretrváva septovaný fluidotorax vľavo, preto konzultovaný hrudníkový chirurg, ktorý odporúča dekortikáciu pľúc. Po štyroch týždňoch hospitalizácie na I. KAIM je celkový stav pacienta stabilný, pacient je pri vedomí s GCS 15 b, na tlakovej podpornej ventilácii, je odpájaný od UPV cez tracheostómiu (netoleruje záťaž), pretrváva oligúria s potrebou pokračovania v MEL, je kardiálne kompenzovaný, pre sínusovú tachykradiu v liečbe ponechaný metoprolol. Postupne pacient dýcha spontánne cez Ayrovo T s následnou dekanyláciou tra-cheostómie. Počas rehabilitačnej liečby pacienta verti-kalizujeme a začína chodiť. Dochádza k miernej úprave azotémie, preto nefrológ neindikuje dlhodobý dia-lyzačný program. Kombinovaná antihypertenzná liečba bola postupne redukovaná. Po šiestich týždňoch hospitalizácie je pacient pri vedomí, dýcha spontánne cez O2 maskuje hemodynamicky kompenzovaný, preto po dohode s chirurgom bol preložený na chirurgickú JIS v stabilizovanom stave. Tab. 1. Laboratórne výsledky pacienta z kazuistiky pred začatím a v priebehu suplementácie selénu Tab. 1. Laboratory results from casuistic research before start selenium supplementation and within therapy Pred podaním selénu 2. deň terapie 7. deň terapie Leukocyty (109/1) 6,6 12,9 20,9 Neutrofily/Lymfocyty 9,9 11 12 Trombocyty (109/1) 233 138 313 Fibrinogen (g/1) 5,76 ň 5,33 Laktát (mmol/1) 1,01 2,24 1,85 CRP (mg/1) 171 163 132 Prokalcitonín (/ig/1) 100 20,3 10,6 GPx (^kat/1) 0,55 2,66 3 GR (/íkaťl) 0,1813 0,3 1.65 Rozhledy v chirurgii 519 18 DISKUSIA Voľné radikály zohrávajú dôležitú úlohu v patogené-ze syndrómu systémovej zápalovej odpovede, sepsy, ťažkej sepsy, septického šoku a akútnej pankreatitídy [5]. Samotné voľné radikály, ako produkt leukocytov majú v prvotnej fáze sepsy dôležitú antimikrobiálnu úlohu pri ničení patogénnych mikroorganizmov. Problémom sa stávajú až vtedy, keď ich tvorba je nekontrolovateľná a ďaleko prevyšuje vychytávaciu schopnosť antioxidantov, a tak dochádza k vzniku oxidačného stresu a aktivácii patologických kaskád [9]. Vznik a účinok voľných radikálov je v rovnováhe s pôsobením antioxidačného obranného systému organizmu [5]. Exhaustná tvorba kyslíkových radikálov indukuje tvorbu cytokínov imunokompetentnými bunkami, ktoré ďalej aktivujú zápalovú kaskádu a vplývajú na ex-presiu adhéznych molekúl na povrchu buniek. Voľné radikály ovplyvňujú kaskádu intercelulárnych dejov, čoho výsledkom je aktivácia génov dôležitých z hľadiska zápalu. Princípom tejto suplementačnej antioxidačnej terapie je ovplyvnenie zápalovej kaskády ešte v počiatku SIRS a tým priaznivo ovplyvniť klinický priebeh [5, 10]. V skorom štádiu ochorenia u pacientov s ťažkou pan-kreatitídou bola experimentálne zistená vysoká tvorba voľných radikálov. Takisto príjem a plazmatické hladiny selénu sú v tejto skupine znížené. V niektorých medicínskych centrách využívajú adjuvantnú terapiu se-lénom pri akútnej pankreatitíde dlhší čas a boli zaznamenané sľubné výsledky. Príkladom je výsledok retrospektívnej štúdie, kde bolo zistené v priebehu desiatich rokov zníženie mortality u pacientov s akútnou pan-kreatitídou z 35 % na 16 % [8]. Pri experimentálnych meraniach hladiny selénu u kriticky chorých pacientov so sepsou a SIRS bol zistený markantný pokles plazmatického selénu a bola zaznamenaná signifikantná negatívna korelácia medzi plazmatickou hladinou selénu, APACHE II a SAPS II [11]. Referenčná hodnota plazmatického selénu je v rozpätí 89-114 Kg/l. Tieto ťažké stavy sa spájajú s presunom vitamínov a stopových prvkov do tkanív, v ktorých sa syntetizujú proteiny teraz v oveľa väčšej miere ako za fyziologických podmienok. Tento presun spôsobuje relatívny deficit cirkulujúcich antioxidantov. Klinické štúdie u pacientov so SIRS, sepsou, ARDS a pankrea-titídou poukazujú na to, že suplementácia selénu priaznivo ovplyvňuje prežívanie [8]. Podávanie selén obsahujúcich doplnkov sa zdá byť prospešným z hľadiska zníženia mortality [5]. Problémom pri tejto terapii je správne načasovanie, odhad dávky, a doby podávania. Suplementácia v neskorých fázach SIRS a ťažkej sepsy sa zdá byť neúčinnou terapiou [12]. Selén je dôležitý mikronutrient. Tvorí nebielkovino-vú súčasťou dôležitého antioxidačného enzýmu gluta-tiónperoxidázy (GPx), od ktorej úzko závisí integrita bunkových a subcelulárnych membrán. Antioxidačné vlastnosti enzýmu sú bytostne závislé od prítomnosti 520 selénu [13]. Prísun vysokých dávok selénu u zdravých ľudí počas dlhého obdobia niekoľkých rokov sa spája s ochorením selenózou. Intoxikácie selénom u kriticky chorých pacientov pozorované doteraz neboli, a to ani pri štúdiách kde podávali vyššie dávky selénu [10]. Dávka 1000 @g/deň podaná intravenózne bola dobre tolerovaná pacientmi na JIS [5]. Dávkovanie selénu pri ťažkej akútnej pankreatitíde nie je striktne odporúčané, preto sme používali priemerné dávkovanie vychádzajúc z výsledkov viacerých randomizovaných štúdií [7,14]. Selén sa uplatňuje ako kofaktor enzýmu glut-hationperoxidázy, ktorý patrí do skupiny enzýmov vykazujúcich peroxidázovú aktivitu, jej hlavnou úlohou je ochrana bunkových štruktúr pred oxidačným poškodením. Biochemická funkcia GPx je redukovať lipidové peroxidy na im korenšpodujúce alkoholy a redukovať voľný peroxid vodíka na vodu. Substrátom pre túto reakciu je glutatión [15, 16]. Suplementáciou glutamínu sa zvyšuje efektivita účinku GPx. Regenerácia GSH z oxidovanej formy GSSG je katalyzovaná glutatión re-duktázou. To že u pacienta z kazuistiky nedochádza k zvýšeniu aktivity GR, je možné vysvetliť z tohto hľadiska, že regenerácia GSSG na GSH nie je zatiaľ nutná, kedže zároveň podávame aj alanylglutamín, pre-kurzor GSH [13]. Priame meranie dynamiky antioxidačných enzýmov u pacienta z kazuistiky poukazuje na ich aktiváciu suple-mentovanými mikronutrientmi. Zároveň je tento jav sprevádzaný poklesom plazmatickej hladiny prokalcitoní-nu, čo je známkou efektívnej terapie sepsy. Taktiež v priebehu liečby dochádza k významnému poklesu hladiny CRP. Laktát sa výrazne nemení. Zvýšená hodnota GPx v siedmy deň od aplikácie prvej dávky selénu a glutamínu svedčí o zvýšenej aktivite antioxidačného systému. Pomer neutrofilov a lymfocytov má priaznivú klesajúcu tendenciu aj napriek stúpajúcej hodnote leukocytov. Pomer neutrofilov a lymfocytov jednoducho a spoľahlivo vystihuje mieru závažnosti oxidačného stresu a systémového zápalu. Bola zistená súvislosť medzi závažnosťou klinického stavu a mierou neutrofílie a lymfocytopénie [17]. Výsledky tejto kazuistiky potvrdzujú priaznivý vplyv su-plementácie selénu na ťažkú formu akútnej pankreatitídy a jej septické komplikácie. ZÁVER Podpora antioxidačných mechanizmov sa zdá byť prínosná pre pacientov s diagnostikovanou ťažkou akútnou pankreatitídou. Načasovanie a dávkovanie antioxidantov a kofaktorov antioxidačných enzýmov je pre terapiu zásadné. Táto špecifickosť dávkovania je veľmi významná najmä pri terapii selénom. Optimálna terapeutická dávka, ku ktorej sme dospeli na podklade výsledkov viacerých štúdií bola určená na 750 mg/deň a doba suplementácie šiestich dní. So suplementáciou je nutné začať ihneď po stanovení diagnózy. Rozhledy v chirurgii 19 Zoznam skratiek: APACHE II Acute Physiology and Chronic Health Evaluation II ARDS akútny syndróm systémovej zápalovej odpovede acute respiratory CVVHD CRP C-reaktívny protein GCS Glasgow coma scale GPx glutatión peroxidáza GSH redukovaná forma glutatiónu GSSG oxidovaná forma glutatiónu MEL mimotelová eliminačná liečba SAPS II Simplified Acute Physiology Score SIRS syndróm systémovej zápalovej odpovede systemic inflammatory response syndrome LITERATÚRA 1. Rau, B M., Krüger, C. M., Hasel, C, Oliveira, V., Rubie, C, Beger, H. G., a kol. Effects of immunosuppressive and immu-nostimulative treatment on pancreatic injury and mortality in severe acute experimental pancreatitis. Pancreas, 2006; č. 33, s. 174-183. 2. Rau, B. M.„ Bothe, A., Kron, M., Beger H. G. Role of early multisystem organ failure as major risk factor for pancreatic infections and death in severe acute pancreatitis. Clin. Gastroenterol. Hepatol., 2006; č. 4: s. 1053-1061. 3. Lakyová L., Toner, Radoňak J., Šimon, R., Toporcer, T., Vajó, J., Belák, J. Spontánna perforácia ductus choledochus pri akútnej pankreatitíde - kazuistika. Rozhl. Chir., 2008, roč. 87, č. 2, s. 92-95. 4. Balthazar, E. J: Staging of acute pancreatitis. Radiologic clinics of North America, 2002; 40 (6): s. 1199-1209. 5. Geoghegan, M., McAuley, D., Eaton, S., Powel-Tuck, J. Selenium in critical illness. Critical Care, 2006, 12, s. 136-141. 6. Nathens, B. A., Curtis, R. J., Beale, J. R., et al. Management of the critically ill patient with severe acute Pankreatitis. Critical Care Medicine, 2004, roč. 32, č. 12, s. 2524-2536. 7. Heyland, D., Dhaliwal, R., Suchner, U., Berger, M. M. Antio- xidant nutrients: a systematic review of trace elements and vitamins in the critically ill patient. Intensive Care Medicine, 2005, roč. 31, str. 327-337. 8. Heyland, D., Dhaliwal, R., Day, A., Drover, J., Cote, H., Wis-chmeyer, P. Optimizing the dose of glutamine dipeptides and antioxidants in critically ill patients: a phase I dose-finding study. J. Parenteral Enteral Nutrition, 2007, roč. 31, s. 109-118. 9. Berger, M., Chiolero, R. Antioxidant supplementation in sepsis and systematic inflammatory response syndrome. Critical Care Medicine, 2007; roč. 35, s. 584-590. 10. Angstwurm, M. W. A., Schottdorf, J., Schopohl, J., Gaertner, R. Selenium replacement in patients with severe systemic inflammatory response syndrome improves clinical outcome. Critical Care Medicine, 1999, roč. 27, s. 1807-1813. 11. Sakr, Y., Reinhart, K., Bloos, F., Marx, G., Russwurm, S., Bauer, M., Brunkhorst, F. Time course and relationship between plasma selenium concentrations, systemic inflammantory response, sepsis, and multiorgan failure. Br. J. Anaesth., 2007, roč. 98, s. 775-784. 12. Forceville, X.,Vitoux, D., Gauzit, R., Combes,A., Lahilaire, P., Chappuis, P, Selenium, systemic immune response syndrome, sepsis, and outcome in critically ill patients. Critical Care Medicine, 1998, roč. 26, s. 1536-1544. 13. Saláma, A., Sakr, Y., Reinhart, K. The role of selenium in critical illness: Basic science and clinical implications. Critical Care Medicine, 2007, roč. 7, s. 127-138. 14. Kuklinski, B., Buchner, M., Schweder, R., Nagel, R.Akute Panc-reatitis-eine „Free Radical Disease: Letalitatssenkung durch Nat-riumselenit (Na2Se03)-Therapie. ZGestame Inn. Med., 1991,46: s. 145-149. 15. Forceville, X. Seleno-enzymes and seleno-compounds: the two faces of selenium. Critical Care, 2006, roč. 10, s. 180. 16. Muller, F. L., Lustgarten, M. S., Jang, Y., Richardson, A., van Remmen, H. Trends in oxidative aging theories. Free Radic. Biol. Med., 2007, roč. 43, s. 477-503. 17. Záhorec, R. Pomer neutrofilov a lymfocytov - rýchly a jednoduchý ukazovateľ systémového zápalu a stresu u pacientov v kritických stavoch; Bratislavské Lekárske Listy, 2001, č. 102: s. 5-14. MUDr. Ladislav Kočan I. klinika anestéziológie a intenzívnej medicíny Trieda SNP 1 040 01 Košice Slovenská republika e-mail: kocanladislav@yahoo.com Rozhledy v chirurgii 521 20 Původní práce Akútne multiorgánové zlyhanie po kolonoskopii s polypektómiou Ladislav Kočan, Janka Vašková, Ladislav Vaško, Lucia Lakyová, Hana Kočanová, Jana Simonová, Robert Simon, Jozef Firment I. klinika anestéziológie a intenzívnej medicíny, Košice, Slovenská republika SOUHRN Východisko. Závažné intraabdominálne infekcie patria medzi život ohrozujúce ochorenia. Vznikajú na podklade šírenia lokalizovaných zápalov alebo narušenia integrity črevnej steny. Metódy a výsledky. Liečebnou stratégiou je chirurgická intervencia, antimikrobiálna terapia, liečba distribučného šoku a cielená nutrične podpora (1). Predpokladá sa, že suplementácia glutamínu a selénu zlepšuje funkciu črevnej bariéry a regeneruje antioxidačnú obranu (2). U pacientky po endoskopickej polypektómii sa rozvinul septický šok s multiorgánovým zlyhávaním a rozvojom katabolizmu s výrazným poklesom albumínu. Origo sepsy zobrazovacími metódami a probatórnou laparotómiou nebolo zistené. Liečbou distribučného šoku, podávaním širokospektrálnych antibiotik, enterálnym a parenterálnym hradením nutrientov došlo k výraznému zlepšeniu zdravotného stavu. V rámci adjuvantnej terapie boli intavenózne supiementované: glutamín v dennej dávke 2 g a pentahydrát seleničitanu sodného v kontinuálnej infúzii v dávke 750 ug/24 hodín v priebehu 6 dní. Počas terapie došlo k poklesu zápalových markerov: C-reaktívny protein, prokalcitonínu, leukocytov, neutrofilov. Došlo k zvýšeniu hladín albumínu. Závěry. Práca poukazuje na terapeutické možnosti pri liečbe septického šoku a možnosťami reverzie katabolickej fázy ochorenia. Kľúčové slová: kolonoskopia, septický šok, multiorgánové zlyhanie, enterálna výživa, parenterálna výživa. SUMMARY Kočan L, Vašková J, Vaško L, Lakyová L, Kočanová H, Simonová J, Simon R, Firment J. Acute multiple organ failure after endoscopic polypectomy Background. Serious intraabdominal infections belong among life treating diseases. They are based on spreading infections from focal sources of inflammation in abdomen or damaged intestinal wall. Methods and results. Treatment strategies are surgical intervention, antimicrobial therapy, distributional shock treatment and accurate nutritional support (1). Glutamine and selenium supplementation may improve intestinal functions and restore antioxidant defence (2). Septic shock with multiple organ failure accompanied by serious catabolism and decrease of albumin had developed in a patient after endoscopic polypectomy. Infection source was not discovered by medical imaging examinations non surgical laparotomy. After distributive shock treatment, wide spectral antibiotics and enteral and parenteral nutrition the patient's health improved. As adjuvant therapy intravenous supplementation was administered: glutamine in daily dose 2g and sodium selenite in continual infusion in daily dose 750 ug over 6 days. During intensive therapy, inflammatory markers decreased: C-reactive protein, procalcitonin, leukocyte count and neutrophils. Albumin levels increased. Conclusions. The paper describes therapeutic options during septic shock treatment and reversion possibilities in the catabolic phase of disease. Key words: colonoscopy, septic shock, multiorgan failure, enteral nutrition, parenteral nutrition. Ko. Čas. Lék. čes. 2012; 151: 568-578 UVOD Ťažká intraabdominálna infekcia ako aj generalizovaná pe-ritonitida sú život ohrozujúce stavy vyžadujúce si okamžitú chirurgickú intervenciu vzhľadom na vysokú mortalitu. Najčastejšou príčinou vzniku komplikovanej intraabdominál-nej infekcie býva divertikulitída, apendicitída, či perforácia gastrointestinálneho traktu (3). Vznik kolitídy po kolonoskopii ADRESA PRO KORESPONDENCI: MUDr. Ladislav Kočan, PhD. I. klinika anestéziológie a intenzívnej medicíny Trieda SNP 1, 041 90 Košice, Slovenská republika e-mail: kocanladislav@yahoo.com je veľmi zriedkavá komplikácia. Býva sprevádzaná tenezma-mi a krvavými hnačkami, objavuje sa najčastejšie do 48 hodín po kolonoskopii, alebo sigmoidoskopii (4). V literatúre sú dokumentované zriedkavé prípady vzniku ischemickej kolitídy po endoskopickom vyšetrení. Ako mechanizmus vzniku sa popisuje nadmerná distenzia hrubého čreva po jeho naplnení plynom pri vyšetrení, čo znižuje prietok krvi črevom z mukózy do serózy, alebo je to mechanický tlak endoskopu po zavedení. Ischémia s nekrózou črevnej steny môže zasiahnuť hlboko do črevnej steny, a porušiť tak bariéru a spôsobiť následnú translokáciu baktérií. Pridružené ko-morbidity u pacienta zhoršujú následne morbiditu a mortalitu (6). Ako jedna z ďalších možných vyvolávajúcich príčin kolitídy po kolonoskopii je používanie glutaraldehydu pri dezifek-cii endoskopu. Po vyšetrení vzniká difúzny edém mukózy čreva, pričom klinická prezentácia a morfologický vzhľad sliznice môže mimikovať ischemickú kolitídu, alebo infekčnú kolitídu 568 Časopis LEkAŔú českých zoi2; isi (12) 21 (5). Hlavnou liečbou je podávanie antibiotík, prednizonu (ste-roidov) a mesalazínu (4, 5). Prognóza pacientov je zvyčajne dobrá, pokiaľ nedôjde k perforácii čreva. Vtedy dochádza k vysokej mortalite vzhľadom na to, že infekcia sa dostane do dutiny brušnej. V nízkom percente prípadov môže tento vnú-trobrušný zdroj infekcie vyústiť až do multiorgánového zlyhania (MOFS) (3). Optimálna liečba a pooperačná starostlivosť je stále kontroverzná. Od šesťdesiatych rokov sa preferuje antibiotická liečba proti gram-negatívnym a anaeróbnym baktériám. Táto kombinácia zvyšuje prežívanie u septických pacientov (7). V rámci intenzívnej starostlivosti je dôležité zvolenie vhodnej nutričnej podpory, jej načasovanie, výber, dávkovanie vhodných nu-trientov a spôsob ich aplikácie. Podporou metabolizmu sa zabezpečia energetické požiadavky ale aj zvýšená potreba už chýbajúcich (resp. vyčerpaných zdrojov) aminokyselín a anti-oxidačných molekúl. So stavom imunosupresie a multiorgánového zlyhania korelujú nízke hladiny glutamínu, preto obzvlášť dôležitú úlohu zohráva práve doplnenie glutamínu a jeho prekurzorov. Taktiež suplementácia selénu kriticky chorým pacientom zlepšuje antioxidačný obranný systém organizmu. Nemenej dôležitú úlohu predstavuje včasná enterálna výživa v boji voči atrofii klkov tenkého čreva a následnej translokácie baktérií z čreva do krvného obehu (8). V prezentovanom prípade ide o zriedkavý prípad vnútro-brušnej infekcie spôsobenej kolitídou v koincidencii s obojstrannou pneumóniou, čo vyústilo až do syndrómu systémovej zápalovej odpovede (SIRS) a popis vlastných skúseností s úspešne zvládnutou pooperačnou liečbou špeciálnymi pa-renterálnymi prípravkami, širokospektrálnym antibiotickým krytím ako aj enterálnou výživou. SOUBOR NEMOCNÝCH A POUŽITE METODY Vykonané terapeutické postupy sú na našej klinike rutinne používané. Pacienti, ktorým bol suplementovaný selén a glutamín, boli zaradení do štúdie Se-AOX schválenej Etickou komisiou Univerzitnej nemocnice L. Pasteura. KAZUISTIKA Žena, 66 rokov, podstúpila ambulantnou formou preventívne kolonoskopické vyšetrenie s polypektómiou v celkovej anestézii. V nasledujúci deň sa rozvinul kolapsový stav s hypotenziou TK 60/40 v domácom prostredí. Pacientka bola prvotne ošetrená posádkou rýchlej lekárskej pomoci. Vzhľadom na cirkulačnú nestabilitu pacientky bola jej podaná katecholamínová podpora (efedrín v bolusovej forme). Následne bola hospitalizovaná v okresnej nemocnici na in- ternom oddelení. Hospitalizácia trvala dva dni. Vstupné laboratórne výsledky poukazovali na systémovú zápalovú reakciu (tab. 1). U pacientky naďalej pretrvávala závažná hypo-tenzia vyžadujúca katecholamínovú podporu. Pre podozrenie na infekciu neznámeho pôvodu bola začatá empirická liečba širokospektrálnymi antibiotikami (Cefotaxim, Metronidazol, Ampicilín-Sulbaktam, Ciprofloxacin) a až následne boli odobraté vzorky krvi na hemokultiváciu. RTG vyšetrenie pľúc odhalilo bilaterálnu bronchopneumóniu, viac vpravo. RTG vyšetrením brucha bol zistený ľahko distendovaný, pneuma-tizovaný žalúdok s primeranou plynatosťou čriev bez jasných známok pneumoperitonea. Konziliárny chirurg vyjadril podozrenie na perforáciu hrubého čreva vzhľadom na predchádzajúcu anamnézu polypektómie. Následne indikoval chirurgickú exploráciu. Pri revízii bolo črevo vitálne, bez známok perforácie a bez prítomnosti voľnej tekutiny. V pooperačnom období sa rozvinula akútna renálna insuficiencia kombinovanej etiológie s nutnosťou zahájenia mimotelovej eliminačnej liečby s pretrvávaním zvýšených zápalových markerov. Zistené kultivácie z odohraných krvných vzoriek nepotvrdili prítomnosť mikroorganizmov. U pacientky postupne dochádzalo k respiračnému zlyhaniu, čo si vyžadovalo zaistiť dýchacie cesty orotracheálnou kanylou a napojiť pacientku na umelú ventiláciu pľúc (UVP). Pre rozvinutý syndróm systémovej zápalovej odpovede (SIRS) bez zisteného infekčného fokusu, bola pacientka preložená na vyššie odborné pracovisko za účelom ďalšieho dodiferencovania klinického nálezu a liečby septického šoku. Po prijme na I. kliniku anestéziológie a intenzívnej medicíny Univerzitnej nemocnice Louisa Pasteura v Košiciach sme znova odobrali krvné vzorky, obsahy brušných drénov a materiál z dolných dýchacích ciest na mikrobiologické vyšetrenie. Eskalovali sme antibiotickú liečbu proti G+ (linezo-lid) a G- (imipenem) a anaeróbnym baktériám (metronidazol), do liečby sme zaradili aj antimykotiká (flukonazol) a imunomodulanciá (polyoxidónium a gamaglobulíny). Realizovali sme echokardiografické vyšetrenie srdca, ktoré vylúčilo infekčnú endokarditídu ako i kontrolné CT vyšetrenie hrudníka a brucha. CT vyšetrenie hrudníka odhalilo in-filtratívne zmeny v ľavom laloku pľúc v hrotovom segmente, v laterobazálnom a posterobazálnom segmente splývali do pomerne homogénneho nevzdušného infiltrátu s pozitívnym bronchogramom. Vpravo bola zvýšená denzita pľúcneho pa-renchýmu v rozsahu celého dolného laloka a čiastočne v zadnom segmente horného laloka. CT vyšetrenie brucha odhalilo malé abscesové ložisko v pravom laloku pečene o veľkosti 0,5 cm, a zväčšený žlčník o šírke 4,25 cm. Intrahepatálne žlčové cesty boli bez dilatácie a a perihe-patálny priestor bol bez prítomnosti voľnej tekutiny. Menšie množstvo voľnej tekutiny bolo zobrazené v perisplenickom a v ľavom subfrenickom priestore, taktiež obojstranne peri- Tab. 1. Dynamický vývoj hematologických a biochemických parametrov počas hospitalizácie v okresnej nemocnici Čas hospitalizácie Začiatok ochorenia 24 hodin 48 hodin leukocyty (109.|-1) 25,8 27,6 29,8 neutrofily (%) 95,5 92,3 93,4 lymfocyty (%) 2,7 3,1 3,5 trombocyty (1031"1) 165 94 69 albumín (g.I"1) 41.4 27,2 25,2 fibrinogén (g.I'1) 1,9 1,11 1,72 urea (mmol/l) 14,4 14,7 21,1 kreatinin (pmol.ľ1) 180 333 447 laktát (mmol.ľ1) 2,9 3,1 3,4 CRP (mg.ľ1) 127,9 210,9 213,2 prokalcitonín (pg.l"1) 100 216 70 AST (ukat.l1) 3,92 5,15 4,12 ALT(ukat.ľ1) 2,2 2,89 3,12 GGT (pkat.l-1) 1,99 1,1 1,13 ČASOPIS LÉKAŘŮ ČESKÝCH 20 I 2; i5i (12) 569 22 Tab. 2. Dynamický vývoj hematologických a biochemických parametrov počas hospitalizácie na I. KAIM 1. deň pred začatím terapie 8. deň 16. deň leukocyty (109.ľ1) 16,94 15,8 12,84 neutratily (%) 94 86 85 lymfocyty (%) 3,8 3,9 3,8 trombocyty (103|-1) 100 170 419 albumin (g.l~1) 22,8 30,1 41,2 fibrinogen (g.l"1) 2.03 2.06 2.12 urea (mmol/l) 25 15,91 15,13 kreatinin (umol.ľ1) 517 235,7 105 laktát (mmol.l-1) 3,6 2,1 2 CRP (mg.l-1) 196 267,1 75 prokalcitonin (pg.l1) 9,55 1,11 0,78 AST (Mkat.ľ1) 3 2,12 1,76 ALT (ukat.r1) 2,6 1,12 1,01 GGT (Mkat.ľ) 1,09 0,89 0,9 renálne, ale bez príznakov abscesu. V pravom hypogastriu, v tesnej blízkosti drénu, bol zreteľný konvolut vzájomne fixovaných črevných kľučiek, ktoré naliehali na brušnú stenu ako aj v ľavom mezogastriu s nálezom konvolutu fixovaných črevných kľučiek svedčiacich pre prítomnosť kotitfdy bez príznakov abscesového ložiska. Konziliárny chirurg vzhľadom na CT nález odporúčal pokračovať v doterajšom konzervatívnom postupe. Pre akútne obličkové poškodenie v štádiu F, podľa klasifikácie RIFLE, bola zahájená mimotelová eliminačná liečba (MEL) formou kontinuálnej veno-venóznej hemodialýzy (CVVHD) iniciálne s citrátovou (Ci-Ca) antikoaguláciou. Pre cirkulačnú instabilitu boli do liečby zaradené katecho-lamíny v kontinuálnej infúzii (noradrenalín 0,5 ug/kg/min). Pacientka bola naďalej napojená na umelú ventiláciu pľúc s režimom tlakovej podpory so zaradením vyšších hodnôt pozitívneho endexpiračného tlaku. Príjem živín bol zabezpečený enterálnou výživou cez nazogastrickú sondu (Nutrison-Multifibre) a podporený parenterálnymi výživovými roztokmi podávanými intravenózne (Smofkabiven a Aminomix II). Výživa bola obohatená o glutamín a jeho prekurzory (ala-nylglutamín do centrálneho venózneho katétra v dennej dávke 2 g). Do liečby boli pridané liečivá s antioxidačnými účinkami. Vitamin C, vitamín E a v kontinuálnej infúzii selén v dennej dávke 750 ug počas šiestich dní (vo forme penta-hydrátu seleničitanu sodného, čo zodpovedá 250 ug selénu na deň). Pre výraznú expektoráciu a opakovaný vzostup zápalových parametrov na základe mikrobiologických výsledkov z dýchacích ciest bola antibiotická liečba deeskalovaná na Cefoperazón/Sulbaktám (Sulperazon) v dávke 2 g každých 8 hodín. Opakované RTG vyšetrenia hrudníka v nasledujúcich dňoch hospitalizácie potvrdili regresiu infiltratívnych zmien. Kontrolné CT vyšetrenie brucha s aplikáciou kontrastnej látky Ultravist 370/100 ml v dvojfázovom režime bolo realizované po siedmich dňoch. V pečeni v pôvodnej lokalizácii pretrvávala pôvodná cystoidná lézia bez signifikantného postkontrastného zvýraznenia a bez evidentnej progresie veľkosti. Perisplenicky, períhepatálne a v malej panve bolo zistené menšie množstvo tekutinových kolekcií. Dutina brušná bola bez známok pneumoperitonea. V porovnaní s predchádzajúcim CT vyšetrením došlo k ústupu prejavov kolití-dy, s pretrvávaním edematóznych prejavov v oblasti steny colon descendes a sigmy. Na laterálnej stene colon des-cendes sa zobrazoval divertikel o priemere 13 mm. Nález na čreve poukazoval na možné reaktívne zmeny pri diver-tikulitíde. Klinický stav pacientky sa postupne zlepšoval, hodnoty zápalových parametrov postupne vykazovali klesajúcu tendenciu, cirkulácia sa stabilizovala, čo umožňovalo postupne znižovať vysoké dávky noradrenalínu až po jeho vysadenie z liečby. Pre pretrvávajúcu renálnu insuficienciu musela byť naďalej vedená MEL formou intermitentnej hemodialýzy s nutnou prísnou tekutinovou bilanciou. Pacientka bola postupne odpájaná od UVP a extubovaná. Pri sporadických poklesoch saturácie krvi kyslíkom bola napojená na dýchací režim neinvazívnej ventilácie pomocou tvárovej masky. Antibiotická liečba bola postupne vysadená. Vzhľadom na iniciálne vysoké dávky noradrenalínu pre cirkulačnú instabilitu ako aj napojenie pacientky na kontinuálnu elimináciu bolo polohovanie pacientky problematické s následnou poruchou celistvosti kože v sakrálnej oblasti, ktorá bola ošetrovaná polyvidonjodidom (Betadine) a kyslíkom. Pacientka bola po dohovore preložená do spádovej nemocnice v zlepšenom zdravotnom stave, s potrebou pokračovania intermitentnej hemodialýzy. DISKUSIA U pacientky z kazuistiky, ktorá podstúpila diagnostickú ko-lonoskopiu s polypektómiou, došlo k rozvinutiu symptómov distribučného šoku, ktorý je charakteristický pre septický šok. Podľa názoru Wellsa a Erlandsena (9), aj keď dostupné sú len nepriame dôkazy, je za rozvojom bakteriémie a sepsy translokácia baktérií z čreva do krvného obehu. Niektoré novšie štúdie (10) však klinický význam translokácie baktérií spochybňujú. Rôntgenovým zobrazením brucha nebola zistená prítomnosť pneumoperitonea, ktoré by poukazovalo na perforáciu v gastrointestinálnom trakte, ale konziliárny chirurg nevylúčil poškodenie črevnej steny a indikoval revíznu laparo-tómiu brucha, nakoľko perforácia hrubého čreva predstavuje závažnú komplikáciu, ktorá môže vyústiť do vzniku sterkorál-nej peritonitídy so septickým šokom. Poškodenie fyziologickej črevnej bariéry umožňuje priľnavosť, alebo internalizáciu črevných baktérií bunkami čreva (9, 10). Už translokácia baktérií alebo toxínov do črevnej steny môže vyvolať SIRS a dysfunkciu vzdialených orgánov tým, že aktivuje črevnú zápalovú odpoveď, aj keď sú baktérie odstránené imunologickými bunkami čreva. Za týchto podmienok sa v čreve masívne tvoria cytokíny a ďalšie prozápalové faktory, čím sa mezenteriálna mikrocirkulácia stáva miestom aktivácie cirkulujúcich neutrofilov (10). Na druhej strane, narušenie intesti-nálnej steny a následná translokácia baktérií je u imunode-ficientných pacientov vyvolaná mnohými faktormi a môže viesť k bakteriémíi, sepse a MOFS (11). Sterkorálna peritonitída však revíziou potvrdená nebola, neboli zachytené mikroorganizmy z odobratých vzoriek krvi, preto sa nedal určiť septický šok ako príčina daného stavu pacienta. Uvažovali sme teda o rozvinutom syndróme zápalovej odpovede s multiorgánovým zlyhávaním, ktorý vznikol na podklade ťažkej bilaterálnej pneumónie, prítomnej divertikulitíde 570 Časopis lékařů českých 20i2; isi (12) 23 a dekompenzácii po celkovej anestézii, ktorý vzniká po uvoľnení prozápalových cytokínov do krvného obehu. Črevo patrí k najcitlivejším a zároveň svojou anatómiou, spektrom funkcií a prebiehajúcimi metabolickými pochodmi k najkomplikovanejším orgánom v tele. Vedľa dôležitej trávia-cej funkcie a absorpcie živín, plní funkciu endokrinného orgánu, ktorý produkuje gastrointestinálne hormóny. Je bohaté na obsah lymfatického tkaniva, má podstatnú funkciu v imunitnom systéme a tvorí vitálne dôležitú veľkoplošnú bariéru medzi vonkajším a vnútorným prostredím v organizme. Terapeutická stratégia ďalšej liečby bola založená na chirurgickom konzervatívnom postupe, a to preplachovaním brušnej dutiny poly-vidonjodidovými roztokmi cez brušné drény, podávaním širo-kospektrálnych intravenóznych antibiotík a voľbou kombinácie enterálnej a parenterálnej výživy. Z hľadiska udržania štrukturálnej a funkčnej integrity črevnej sliznice je preferovaná en-terálna výživa. Doplnková parenterálna výživa je vhodná na substitúciu tých živín, ktoré sa obtiažne suplementujú enterál-nou formou. Vplyvom parenterálnej výživy bohatej na aminokyseliny sa ovplyvňujú proteosyntetické procesy, čo má pozitívny vplyv na bilanciu dusíka. Odporúčaná dávka aminokyselín je 1,5 g/kg telesnej hmotnosti za deň. Toto množstvo by malo pozitívne ovplyvniť endogénnu proteosyntézu, kým na úroveň katabolizmu nemá zásadný vplyv. Kriticky chorí pacienti majú ťažký deficit glutamínu. Koncentrácia glutamínu výrazne klesá pri ťažkých katabolických stavoch, ako sú veľké traumy, ťažká sepsa, veľké operačné výkony. Výrazný úbytok glutamínu sa prejavuje zhoršením regeneračných a imunitných procesov, ako aj narušením integrity črevnej bariéry. Ten je významným zdrojom energie pre enterocyty a imunokompetentné bunky. Nízke hladiny glutamínu korelujú so vznikom multiorgánové-ho zlyhania. Suplementácia glutamínu jednoznačne zlepšuje antioxidačný stav pacienta, znižuje výskyt infekčných komplikácií a skracuje dobu hospitalizácie. Pre nestabilitu sa v roztokoch pripravuje zakomponovaný vo forme dipeptidov, ako je napríklad kombinácia glutamín-alanín alebo glutamín-glycín (11,12). Počiatočná fáza metabolizmu glutamínu je rovnaká pre všetky imunitné bunky, syntetizuje sa v nej redukovaný nikotínamidadeníndinukleotidfosfát (NADPH) a arginín. Čím sa vysvetľujú aj vysoké nároky na množstvo glutamínu a samotnú účinnosť týchto buniek obzvlášť u ťažko chorých, ktoré práve NADPH využívajú na tvorbu aktívnych zlúčenín voči mikroorganizmom. NADPH vzniká z malátu pri premene na pyruvát a je východiskovou látkou pre syntézu superoxidové-ho radikálu NADPH oxidázou obzvlášť u fagocytujúcich buniek, ktorými sú neutrofily, makrofágy ako aj lymfocyty, a v kombinácii s reakciou katalyzovanou myeloperoxidázou vytvára najsilnejší fyziologický oxidant a mikrobicíd, kyselinu chlórnu. Z arginínu vzniká účinkom syntázy oxidu dusného NO, ktorý vytvára so superoxidom ďalšiu účinnú látku peroxi-nitril. Aplikácia aj malého množstva glutamínu výrazne napomáha udržiavať bariérovú funkciu čreva, indukuje tvorbu imu-noglobulínovv čreve a zlepšuje perfúziu splanchnickej oblasti (12). Príčinou vzniku lymfocytopénie u pacientky z kazuistiky môže byť reakcia na vysoké hladiny katecholamínov, kortizolu a prolaktínu pri záťažových situáciách, ako je ich marginácia v RES (retikulo-endotelový systém) tkanivách, lymfatických štruktúrach a slizničnom imunitnom systéme asociovanom s črevom (GALT) alebo ich urýchlená apoptóza. Príčiny ne-utrofílie sú viaceré. Hypotézy založené na experimentálnych výsledkoch poukazujú na možnosť zvýšenia počtu neutrofi-lov ich demargináciou z cievnej steny ako aj indukciou tvorby zápalovými mediátormi (13). U pacientky došlo počas terapie k úprave trombocytopénie, ktorá je častým nálezom u septických pacientov. Pokles množstva trombocytov u septických pacientov pod 150 x 109 ľ1 sa vyskytuje v 35^14 %. Mechanizmus trombocytopénie u septických pacientov je multifaktoriálny (14). Úprava trombocytov a populácií bielych krviniek do referenčného rozmedzia svedčí pre pokles zápalovej odpovede (15). Aj zvýšený prísun selenu zvyšuje antioxidačnú ochranu a napomáha zvládnuť oxidačný stres pri SIRS, a to dvojako, prooxidačným účinkom pred zabudovaním a výrazne po zabudovaní do selenoproteínov. Môže tým zvýšiť prežívanie pacientov so sepsou (9). Podľa me-ta-analýzy Finley (1999) môžu byť aj trombocyty ukazovateľom adekvátneho príjmu selénu. V priebehu 7 dní po zahájení liečby (tab. 2), ich početnosť výrazne stúpol (16). U kriticky chorých pacientov sa črevo považuje nielen za cieľové miesto pôsobenia, ale aj za miesto produkcie mediá-torov zápalu, ktoré prispievajú k vzniku SIRS ako aj bakte-riémie, sepsy alebo septického šoku s multiorgánovým zlyhávaním. Črevo sa môže stať zdrojom zápalových mediátorov. Nebakteriálne faktory produkované v čreve a nachádzajúce sa v mezenteriálnej lymfe vedú následne k poškodeniu vzdialenejších orgánov. Táto interpretácia úlohy čreva ako hlavného orgánu zodpovedného za produkciu zápalových mediátorov SIRS a pre vznik MOFS (17) sa teda javí ako vhodné vysvetlenie aj napriek negatívnym výsledkom hemokultivácie. Keďže pacientke bola nasadená empirická liečba široko-spektrálnymi antibiotikami už v periférnej nemocnici, prítomnosť mikroorganizmov skutočne nemusela byť preukázaná. Aj na základe vysokých hodnôt prokalcitonínu (PCT) (viď tab. 1) predpokladáme generalizovanú infekciu, ktorú organizmus zvládol, čo potvrdzuje aj vývoj hodnôt C-reaktívneho proteinu (CRP) (viď tab. 2). Poškodenie pečene sa prejavilo poklesom syntetickej činnosti a to znížením albumínu a fibrinogénu. Silný pokles albumínu mohol participovať na výskyte tekutín v brušnej dutine zistených pri CT vyšetrení. Podporná liečba, prísun aminokyselín a ďalších živín enterálnou a parenterálnou výživou zabezpečili obnovu funkcií pečene, čo sa prejavilo návratom uvedených bielkovín do rozmedzia normálnych hodnôt. Aktivity enzýmov ALT, AST, GGT tiež poklesli, ale ich zvýšenie nemusí odzrkadliť len poškodenie pečene, nakoľko nie sú orgánovo špecifické. Pokles hodnôt urey a kreatininu sú vyvolané hemodialýzou, ale poukazujú na stále nedostatočnú funkčnosť obličiek. ZÁVER Zvládnutie septického šoku je komplexný problém vyžadujúci si multidisciplinárny prístup. Základom liečby je nájdenie origa infekcie a cielená chirurgická liečba v prípade vnútro-brušnej infekcie. Pri rozvoji MOFS si vyžaduje kritický stav pacienta nielen širokospektrálnu ATB liečbu, parenterálnu a enterálnu výživu. V ťažkom katabolickom stave a oxidačnom strese prináša benefit aj suplementácia glutamínu a selénu. Udržiava sa tým bariérová funkcia čreva, zlepšuje perfúzia splanchnickej oblasti, indukuje sa tvorba imunoglo-bulínov v čreve a podporí sa antioxidačná obrana organizmu, čo zvyšuje pravdepodobnosť prežitia pacienta. Skratky ATB - antibiotiká Ci-Ca - citrátový - kalciový modul CRP - C-reaktivny protein CT - počítačová tomografia CVVHD - kontinuálna veno-venózna hemodialýza GALT - s črevom asociovaný slizničný imunitný systém MEL - mimotelová eliminačná liečba MOFS - syndróm multiorgánoveho zlyhania NADPH - redukovaný nikotínamidadeníndinukleotidfosfát NO - oxid dusnatý PCT - prokalcitonín RES - retikulárny endoteliálny systém RIFLE - klasifikačný systém pre akútne poškodenie obličiek SIRS - syndróm systémovej zápalovej odpovede UVP - umelá ventilácia pľúc časopis lékařů českých 2012: 151 (12) 571 24 LITERATURA 1. Záhorec R, Firment J, Straková J, Mikula J, Malík P, Novák I, Zeman J, Chlebo P. Epidemiology of severe sepsis in intensive care units in the Slovak Republic. Infection 2005; 33(3): 122-128. 2. Andrews P, Avenell A, Noble D, Campbell M, Croal B, Simpson W, Vale L, Battison C, Jenkinson D, Cook J. 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Ťažký priebeh Stillovej choroby s multiorgánovým zlyhávaním a závažnou pečeňovou dysfunkciou. Anest intenziv Med 2011; 22(6): 337-342. 9. Wells CL, Erlandsen SL. Bacterial translocation: Intestinal epithelial permeability. In: Rombeau JL, Takala J. (eds.) Gut Dysfunction in critical illness. Berlin: Springer-Verlag 1996; 131-149. 10. Deitch EA. Bacterial translocation or lymphatic drainage of toxic products from the gut: What is important in human beings? Surgery 2002; 131: 241-244. 11. Zadák Z. Umělá výživa při poškození čreva. In: Zadák Z. Výživa v intenzivní péči. Praha: Grada Publishing 2008; 383-391. 12. Páchl J, Roubík K. Základy anesteziologie a resuscitační péče dospělých i dětí. Praha: Karolinum 2005; 253-267. 13. Zahorec R. Ratio of neutrophil to lymphocyte counts-rapid and simple parameter of systemic inflammation and stress in critically ill. Bratisl Lek Listy 2001; 102(1): 5-14. 14. Kirschenbaum L, Mckevitt D, Rullan M, Reisbeck B, Fujii T. Importance of platelets and fibrinogen in neutrophil-endothelial cell interactions in septic shock. Critical Care Medicine 2004; 32: 1904-1909. 15. Spaková T, Rosocha J, Lacko M, Harvanová D, Gharaibeh A. Treatment of knee joint osteoarthritis with autologous platelet-rich plasma in comparison with hyaluronic acid. Am J Phys Med Rehabil 2012; 91(5): 411-417. 16 Finley JW, Duffield A, Ha P, Vanderpool RA, Thomson CD. Selenium supplementation affects the retention of stable isotopes of selenium in human subjects consuming diets low in selenium. Br J Nutr 1999; 82: 357-360. 17. De-Souza DA, Greene LJ. Intestinal permeability and systemic infections in critically ill patients: Effect of glutamine. Crit Care Med 2005; 33: 1125-1135. 572 Časopis lěkaRu Českých 20I2: isi (12) 25 r KAZUISTIKA Ťažký priebeh StiHovej choroby s multiorgánovým zlyhávaním a závažnou pečeňovou dysfunkciou Kočan Ladislav1, Vašková Janka4, Vaško Ladislav4, Hoková Hana3, Majerník Miloš1, Krištofova Beáta2, Simonová Jana1, Firment Jozef1 1I. klinika anestéziológie a intenzívnej medicíny, UN LP, Košice, SR 2|. interná klinika, UN LP, Košice, SR 3Klinika anestéziológie, resuscitácie a intenzívnej medicíny ÚVN SNP Ružomberok - FN, SR 4Ústav lekárskej chémie, biochémie a klinickej biochémie, LF UPJŠ, Košice, SR SÚHRN Stillova choroba je ochorenie neznámej etiológie a patogenézy. Je charakterizovaná febrilitami, artritídou, exantémom a možným postihnutím ďalších orgánov. Kazuistika sa zaoberá popisom akútnej exacerbácie a liečby Sti Hovej choroby u 31-ročnej pacientky dlhodobo liečenej kortikoidmi. Pacientka bola prijatá na I. kliniku anestéziológie a intenzívnej medicíny pre febrility do 40 C. s kvantitatívnou poruchou vedomia (sopor) a s prejavmi multiorgánového zlyhávania (pečene, akútneho zlyhávania obličiek, kardiovaskulárneho systému, dýchania a disseminovanej intravaskulárnej koagulopatie). Komplexnými vyšetreniami sa však zdroj sepsy, malignita, hematologické ochorenia a ani infekčné agens nezistili (opakované vyšetrenia vzoriek krvi boli negatívne). Stav bol definovaný ako akútna exacerbácia Stillovej choroby dospelých v. s. komplikovaná syndrómom nadmernej aktivácie makrofágov so sekundárnym multiorgánovým zlyhaním. Do liečby bolo zahrnuté podávanie pulznej kortikoterapie, hepatoprotektív, doplnkovej umelej výživy vrátane parenterálnej suplementácie selénu, širokospektrálnych antibiotík (ATB), umelej ventilácie pľúc a mimotelovej eliminačnej liečby, v ktorej sa po stabilizácii stavu pokračovalo na Internej klinike. Komplexný terapeutický prístup viedol k významnej regresii hepatálnej insuficiencie. Pacientka bola po 28 dňoch hospitalizácie vo významne zlepšenom stave prepustená do ambulantnej starostlivosti. Kľúčové slová: Stillova choroba - syndróm systémovej zápalovej odpovede - selén - enterálna výživa - pa-renterálna výživa - glutatión peroxidáza Abstract Severe course of Still's disease with multiple organ failure with predominant liver failure Still's Disease is a disease of unknown aetiology and pathogenesis, it is characterized by fever, arthritis, salmon-coloured rash and organ failure in severe cases. This is a case report of a 31-year old female patient on long-term steroid treatment who suffered from adult Still's disease. She was admitted to the Clinic of Anesthesiology and Intensive Care with fever up to 40 °C, unconsciousness and signs of multi-organ failure (liver, acute kidney failure, cardiovascular system, lungs, and disseminated intravascular coagulation). A complex of examinations did not confirm the cause to be sepsis, malignancy, a haematological disease or an infectious disease (repeated blood cultures were negative). We concluded this was an acute exacerbation of adult Still's disease complicated by the syndrome of oversize macrophage activation with secondary multi-organ failure.The patient's condition required pulse steroid therapy, hepato-protective therapy and supplementary nutrition inclusive of parenteral selenium supplementation, antibiotics, mechanical ventilation and extra-corporeal elimination therapy. The therapy continued at the Clinic of Internal Medicine. The complex therapy resulted in great regression of the liver insufficiency. After 28 days of hospitalization the patient was discharged in an improved condition. Keywords: Still's disease - systemic inflammatory response syndrome - selenium - enteral nutrition - parenteral nutrition - glutathione peroxidase Anest. intenziv. Med., 22, 2011, č. 6, s. 337-342 Anesteziologie a intenzivní medicína 337 26 Úvod Kriticky chorí pacienti sú zatažení dysfunkciou rôznych orgánových systémov. Rozdielnosti v metabolických pochodoch si vyžadujú specifický terapeutický prístup adekvátne reagujúci na metabolické požiadavky organizmu. Existencia širokého spektra enterálnych a parenterálnych nutričných prípravkov poskytuje účinné terapeutické možnosti zvrátenia nepriaznivej metabolickej situácie. Obzvlášť závažná situácia nastáva pri stavoch spojených so zlyhávaním pečene. Ide o chorobné stavy, akými sú napríklad systémové, he-matologické ochorenia, sepsa a Stillova choroba. Stil-lova choroba dospelých je akútny febrilný syndróm dospelých, typicky postihujúci niekoľko orgánov s klinickými a laboratórnymi abnormalitami, pričom k plnému rozvoju príznakov môže dôjsť až v priebehu niekoľkých týždňov či mesiacov. Stanovenie diagnózy je obtiažne a nasleduje až po vylúčení ostatných príčin systémových prejavov, ako sú horúčky, vyrážky, hepato-splenomegália, lymfadenopatia a sérozitída. Artritída, ktorá sa môže objaviť kedykoľvek v priebehu ochorenia, má rôzny charakter. Asi v polovici prípadov prebieha ako ťažká, deštruktívna polyartritída. Medzi ostatné príčiny systémových prejavov patria infekčné a systémové afekcie a malignita. Liečba spočíva v podávaní nesteroidných antireumatík, glukokortikoi-dov, liekov modiťikujúcich ochorenie a u ťažších ťoriem je indikovaná aj biologická liečba.Toto ochorenie bolo po prvýkrát popísané anglickým pediatrom Ericom By-watersem v r. 1971 [1], ktorý zaznamenal 14 prípadov Stillovej choroby. Dôležitou úlohu v patogenéze chorobných stavov u kriticky chorých pacientov (syndróm systémovej zápalovej odpovede, sepsa a hepatálne zlyhávanie) zohrávajú voľné kyslíkové radikály a reaktívne kyslíkové častice (ROS, reactive oxygen species). Za normálnych okolností vzniká superoxidový radikál (02") mo-novalentnou redukciou molekulárneho kyslíka v každom živom systéme, najmä v reakciách spojených s mitochondriálnym elektrónovým transportom. Diz-mutáciou 02." sa tvorí peroxid vodíka (H202), ktorý nie je voľným radikálom, avšak vedie k tvorbe hydroxylo-vých radikálov (.OH), ktoré sú považované vôbec za najreaktívnejšie [2]. Produkcia ROS však v organizme môže byt zámerná a mohutná ako v prípade aktivácie neutrofilov a je súčasťou antimikrobiálnej ochrany. Ovplyvnenie ale aj zvládnutie oxidačného stresu je teda významné z hľadiska ovplyvnenia patofyziologie daných ochorení [3]. Parenterálne sa na podporu antioxidačného systému využíva suplementácia selénu [4], ktorý je kofakto-rom antioxidačného enzýmu glutatión peroxidázy (GPx). Mnohé štúdie potvrdzujú priamu súvislosť medzi hladinou selénu v plazme, selenoenzýmov a závažnosťou septického stavu [5]. Etiológia Stillovej choroby je dosiaľ neznáma. Najpravdepodobnejšou hypotézou je prehnaná reakcia organizmu na infekčné agens alebo určitý toxický substrát komplexnou interakciou. Popi- 338 sovaný prípad približuje úspešnosť zvolenej terapie pri Stillovej chorobe komplikovanej systémovým prejavom zápalu spojeným zo zlyhávaním pečene. Metodika Vykonané terapeutické postupy sú na našej klinike rutinne používané. Pacienti, ktorým boli stanovené aktivity antioxidačných enzýmov, boli zaradení do štúdie Se-AOX schválenej Etickou komisiou Univerzitnej nemocnice L. Pasteura. Kazu isti ka Pacientka vo veku 31 rokov s dlhodobou imunosu-presívnou liečbou kortikoidmi a s dokumentovanou Stillovou chorobou bola hospitalizovaná pre vertebro-algický syndróm v cervikálnej a lumbosakrálnej oblasti na neurologickom oddelení okresnej nemocnice. Priebeh hospitalizácie bol komplikovaný febrilitami a rozvojom šokového stavu, syndrómom systémovej zápalovej odpovede (SIRS) nejasnej etiológie a multiorgánovým zlyhávaním. U pacientky došlo k rozvoju diseminovanej intravaskulárnej koagulopa-tie (DIC). Pre zhoršujúci sa stav bola pacientka preložená na I. kliniku anestéziológie a intenzívnej medicíny (I. KAIM) Univerzitnej nemocnice L. Pasteura v Košiciach za účelom ďalšej intenzívnej terapie. Ihneď po prijatí na KAIM sa realizovala adjuvantná antioxidač-ná terapia kontinuálnou suplementáciou selénu v dávke 750 ug/24 hodín počas nasledujúcich šiestich dní. Výživa bola zabezpečená podávaním nutrientov enterálnym i parenterálnym prístupom. Nutrične prípravky obsahovali vyšší podiel rozvetvených esenciálnych aminokyselín (valín, leucín, izoleucín) a pre-kurzorov glutatiónu (cystein, glutamín). Pravidelne boli merané aktivity antioxidačných enzýmov, ako aj štandardné zápalové parametre (tab. 1). Bola začatá oxy-genoterapia a empirická kombinovaná širokospektrál-na antimikrobiálna liečba. Pre cirkulačnú instabilitu si stav vyžiadal vazopresorickú podporu noradrenalínom v dávke 0,06 ug . kg-1 . min-1. V laboratórnom skrínin-gu pri prijatí dominovali elevované aminotransferázy, hyperbilirubinemia, zvýšená hladina urey v sére, anémia, leukocytóza, hypokoagulačný stav s trom-bocytopéniou v rámci DIC a zvýšené zápalové mar-kery C-reaktívny protein (CRP) a prokalcitonín (PCT) - viď tabuľka 1. Po konzultácii s hematológom bola realizovaná substitučná hemoterapia. Vzhľadom na rozvíjajúcu sa oligúriu, po konzultácii s nefrológom, bola indikovaná mimotelová eliminačná liečba (MEL) formou kontinuálnej veno-venóznej hemodialýzy (CVVHD, Ci-Ca modul). Do terapie boli zaradené he-patoprotektíva a vitamínová liečba. Po konzultácii s internistom stav hodnotený ako exacerbácia Stillovej choroby a následne aplikovaná pulzná liečba metyl-prednisolonom (iniciálne 500 mg i. v. počas 4 dní). Na 3. deň hospitalizácie bola pacientka pre progresiu res- Anesteziologie a intenzivní medicína 27 Tabulka 1. Laboratórne hodnoty počas hospitalizácii na I. klinike anestéziológie a intenzívnej medicíny Pri prijatí 2. deň terapie 6. deň terapie Leukocyty (109/l) 13,05 12,77 8,54 Neutrofily/Lymfocyty (109/l) 10 11,5 13 Trombocyty (109/l) 41 45 68 Albumín (g/l) 30,7 29,2 30,9 Celkové bielkoviny (g/l) 51,6 48,4 50,8 Fibrinogen (g/l) 0,15 0,87 1,07 ALT (ukat/l) 69.1 22,4 6,44 AST (ukat/l) 173,1 22,4 1,65 Bilirubin celkový (mmol/l) 31 49,3 37,6 Kreatinin (umol/l) 261.5 245,9 194,4 Urea (mmol/l) 11,34 8,56 10,52 Laktát (mmol/l) 7,16 3,15 2,31 CRP (mg/l) 78,6 39,08 4,77 Prokalcitonín (pg/l) 25,66 2.3 0,866 Protrombínový čas (%) 19 41 95 APTT (s) 54,3 41,9 30,2 GPx (ukat/l) 0,1166 0,1912 0,27 GR (ukat/l) 0,2666 0,4122 0,6833 SOD (U/ml) 3,975 4,112 4,450 piračnej insuficiencie intubovaná a prechodne napojená na umelú ventiláciu pľúc (UVP). Boli realizované pomocné zobrazovacie vyšetrenia v rámci detekcie zdroja ťažkej sepsy. CT vyšetrenie brucha nevylúčilo kolitídu v cékoascendentnej oblasti a v oblasti pravého colon transversum. Zároveň bolo prítomné edema-tózne presiaknutie oboch obličiek. CT vyšetrenie pľúc potvrdilo obojstranný fluidotorax a infiltratívne zmeny v dorzobazálnych častiach oboch pľúcnych krídel. Pre meningeálny syndróm a kvadruspasticitu bola realizovaná lumbálna punkcia, vyšetrenie likvoru však bolo negatívne. Po aplikovanej liečbe došlo k parciálnemu zlepšeniu laboratórnych parametrov. Nadálej pretrvávala anúria, boli nutné denné nefrologické konzília pre vyžadujúcu MEL, postupne z CVVHD zmenenej na in-termitentnú hemodialýzu. Po týždni hospitalizácie na I. KAIM bol pacientkin celkový zdravotný stav zlepšený, pacientka bola afebrilná, so spontánnym dýchaním, parametre acidobázickej rovnováhy boli v norme, preto bola extubovaná, hemodynamicky stabilná bez nutnosti pokračovania v katecholamínovej liečbe. ATB liečba bola ukončená, antimykotikum ponechané len v profylaktickej dávke. Nutrícia bola nadalej zabezpečená kombináciou enterálnej a parenterálnej výživy. Tabuľka 2. Laboratórne hodnoty počas hospitalizácie I. klinike internej medicíny Pri prijatí Pri prepustení Leukocyty (109/l) norma norma ALT (ukat/l) 4,54 1,67 AST (ukat/l) 1,35 0,67 GMT (ukat/l) 9,68 6,9 ALP (ukat/l) 1,78 1,48 Kreatinín (umol/l) 374,4 norma Urea (mmol/l) 21,12 norma CRP (mg/l) Norma norma Po šiestich dňoch bolo parenterálne podávanie selénu ukončené. Pacientku sme v stabilizovanom stave preložili na jednotku intenzívnej starostlivosti I. internej kliniky U N LP. Pri príjme na internú kliniku bola pacientka pri vedomí, afebrilná, bez prejavov respiračnej insuficiencie, hemodynamicky stabilná, nadálej pretrvávala anúria. Výsledky vstupných laboratórnych vyšetrení sú uvedené v tabuľke 2. Realizované kompletné mikrobiologické vyšetrenia vrátane hemokultúr boli negatívne. Pre akútne obličkové zlyhanie sme nadálej pokračovali v mimotelovej eliminačnej liečbe, s postupnou úpravou kreatininu na 135,9 umol . H. Taktiež sme pokračovali v pulznej kortikoterapii (v dávke 250 mg metylprednisolonu na deň) s postupnou redukciou dávky až prechodom na pero-rálnu liečbu prednisonom v dávke 40 mg na deň. Pacientke bol odstránený centrálny venózny katéter, nutrícia bola zabezpečená už len enterálnou formou. Ane-mický syndróm sme korigovali krvnými prevodmi. Vzhľadom na postihnutie pečene bola pacientke počas hospitalizácie podávaná hepatoprotektívna liečba zahájená už na KAIM, pri ktorej došlo k parciálnemu poklesu aminotransferáz (tab. 2). Stav pacientky sa po celkovo 28 dňoch hospitalizácie výrazne zlepšil a pacientku sme prepustili do ambulantnej starostlivosti. Diskusia Exacerbácia Stillovej choroby u pacientky bola vyvolaná neznámou príčinou. Infekčná etiológía nebola potvrdená, po diagnostikovaní kritérií SIRS a odobratí hemokultúr boli preventívne podávané širokospektrál-ne antibiotiká. Laboratórne boli po príjme potvrdené známky rozvíjajúceho sa hepatálneho zlyhávania, enormne zvýšené aktivity hepatálnych enzýmov, pokles koagulačných parametrov a opakovaný sklon k hypoglykémiam. Stav si vyžiadal podávanie pulznej kortikoterapie, hepatoprotektív, umelú pľúcnu ventiláciu, mimotelovú eliminačnú liečbu a špecifickú nutrič-nú podporu, formou enterálnej a parenterálnej výživy rešpektujúcej hepatálne poškodenie. Dôležitou úlohou terapie je ovplyvnenie SIRS, kedy dochádza k masívnej produkcii voľných kyslíkových radikálov (ROS) v organizme. ROS zohrávajú významnú amplifikačnú úlohu v úvodnej fáze SIRS. Ak produkcia reaktívnych foriem kyslíka daleko prevyšuje ich vychytávanie, dochádza k spúšťaniu kaskád imunitných a zápalových dejov, súborne označovaných ako oxidačný stres [6], Ovplyvniť oxidačný stres podporou metabolizmu závisí aj od optimálne zvolenej parenterálnej a enterálnej výživy, ktorá okrem iného aktivuje antioxidačnú obranu organizmu a doplní chýbajúce Anesteziologie a intenzivní medicína 339 28 stopové prvky a spotrebované zásoby vitamínov. U kriticky chorých sa zdá byt' obzvlášť prospešná pa-renterálna suplementácia selénu ako aj prekuzorov glutatiónu. Štúdie potvrdili zlepšenie klinických výsledkov pri podávaní selénu, ktorý napomáhal zlepšovaniu zdravotného stavu pacientov pri infekciách a orgánovom zlyhaní. Pri podávaní glutamínu v rozsiahlych štúdiách sa potvrdilo zníženie infekčných komplikácií u kriticky chorých pacientov [7]. Pečeň je ústredným metabolickým orgánom s ex-krečnými, metabolickými, detoxikačnými, hematologic-kými, hemostatickými funkciami a radou ďalších dôležitých funkcií. V začiatočnej fáze hepatálneho zlyhania dochádza k vyššiemu metabolizmu proteínov s prevahou katabolických dejov. Ústrednú úlohu pri týchto dejoch zohrávajú katabolické hormóny. Prvotným in-zultom zodpovedným za nasledujúce deje sú noxy rôznej etiológie, ako napríklad infekcie, sepsa a vplyv rôznych toxínov. Dochádza k akcelerácii zápalovej odpovede, do obehu sa uvoľňujú mediátory zápalu a zvyšuje sa tvorba proteínov akútnej fázy. Táto fáza je do určitej miery reverzibilná, za podmienky dostatočných kompenzačných mechanizmov, zahŕňajúcich imunitné, metabolické a terapeutické faktory. Umelá výživa u kriticky chorých je limitovaná funkčnosťou gastrointestinálneho traktu z hľadiska voľby výživy enterálnej, doplnkovej alebo parenterálnej. Druhou limitáciou je utilizácia živín v pečeni, ktorá pri he-patálnom zlyhávaní môže byť nedostatočná a je nevyhnutné podávať synteticky pripravené základné stavebné makromolekuly živín. Orgánovo špecifická výživa pri hepatálnom zlyhávaní je založená na využití anaplerotických (AS) a špecificky nutričných substrátov (ŠS) [8]. Narušením jednej čiastkovej reakcie dochádza k zastaveniu celej metabolickej cesty. Suple-mentáciou AS sa doplní substrát za prerušenou reakciou, čím môže sled biochemických reakcií ďalej pokračovať. Suplementácia ŠS za daných podmienok je výhodná z hľadiska lepšej utilizácie a zlepšenia energetickej rovnováhy [8]. Výhodnou alternatívou sa zdá byť výživa doplnková. Dôležitým nutrientom, ktorý by mala obsahovať enterálna výživa, sú mastné kyseliny. Predstavujú významný zdroj pre regeneráciu hepato-cytov. Vhodné je aj podávanie prebiotík (laktulózy), respektive probiotík v rámci enterálnej výživy, z dôvodu zvrátenia hnilobných procesov v čreve na kvasné. Pri parenterálnej výžive je vhodné pacientom podávať 40-45 % rozvetvených aminokyselín, ako aj vyššie množstvá esenciálnych aminokyselín s výnim -kou aromatických, ktorých príjem je potrebné redukovať [9], Za priaznivých podmienok dochádza k obnoveniu fyziologických funkcií pečeňového tkaniva a dochádza k reparácii narušených pečeňových lalôčikov ad inte-grum. Pri nadmernom pôsobení noxy, môže táto fáza prekročiť kompenzačné limity a stať sa ireverzibilnou a vyústiť do fulminantného hepatálneho zlyhania [8]. Dôležitú úlohu v tomto procese zohrávajú aktivované Kupfferove bunky. Sú to mononukleárne makrofágy, ktoré pod vplyvom noxy uvoľňujú enormné množstvo 340 voľných radikálov (ROS) do svojho okolia.Tvorba ROS za fyziologických podmienok prebieha kontinuálne v rámci aeróbneho metabolizmu. ROS slúžia podobne ako aj v iných tkanivách na elimináciu baktérií a sú spúšťačom uvoľňovania signálnych molekúl. Tkanivá sú voči ROS chránené antioxidačným systémom. ROS v pečeni účinkujú na všetky dôležité biomolekuly, proteiny, lipidy a nukleové kyseliny. Poškodenie bunkových membrán hepatocytov je spôsobené ich lipoper-oxidáciou ako aj sekundárnym poškodením vznikajúcimi produktami lipoperoxidácie. ROS sú zodpovedné za zvýšenie aktivity fosfolipázy-A2, čím dochádza k štepeniu fosfolipidov bunkových membrán a poruchám intracelulámej homeostázy Ca2+ [9]. Peroxidované lipidy sú následne rozložené na aldehydy, ketóny, alkoholy a laktóny, ktoré samotné majú toxické účinky. Dôležité je spomenúť rozklad hemoglobínu v pečeni, ktorý môže proces lipoperoxidácie iniciovať. Uvoľnená skupina hému, ktorá obsahuje ióny železa má katalytický účinok na tvorbu peroxidov. Podľa laboratórnych výsledkov dochádzalo k zvýšenej dekompozícii hemoglobínu, čím sa zvýšil aj obsah he-moproteínov, respektive iónov kovov, ktoré proces pe-roxidácie organických substrátov urýchľovali. Zvýšenie aminotransferáz, bilirubinu, zníženie albumínov potvrdzuje poškodenie pečene. Okrem klinických príznakov poškodenia obličiek to potvrdzujú aj zvýšenia kreatininu a močoviny v krvi. Pritom nie je možné vylúčiť ani postihnutie svalov, nakoľko aminotransferázy sú súčasťou každej bunky včítane svalov a na zvýšení hodnôt sa mohli podieľať aj svalové aminotransferázy. Podobne kreatinin vzniká vo svaloch odbúravaním kreatínfosfátu, ale na zvýšení kreatininu v sére môže pôsobiť znížené vylučovanie obličkami. Spomínané látky sa ale dialýzou odstraňujú. Za fyziologických okolností je glykogén uskladnený v pečeni ľahko mobilizovateľný, a je preto dôležitým zdrojom glukózy pre ostatné orgánové systémy. Narušením funkcie glykogenfosforylázy dochádza k poklesu jeho premeny na D-glukózu. Pacienti s hepa-tálnym zlyhávaním sú preto náchylní na hypoglyké-miu. Dochádza k poklesu proteosyntézy, čo sa prejavuje znížením hladín celkových bielkovín a dochádza najmä k poklesu plazmatického albumínu, prealbumí-nu, transferínu, transkortínu a retinol-viažúceho proteinu [9]. U pacientky z kazuistiky boli zaznamenané znížené hladiny celkových bielkovín a najmä albumínu v krvi počas hospitalizácie na I. KAIM. Ďalej dochádza pri hepatálnom zlyhaní k výraznému zvýšeniu plazmatickej koncentrácie aromatických kyselín feny-lalanínu, tyrozínu a tryptofánu, ktoré sú za fyziologických podmienok metabolizované prevažne v pečeni. Narušenie metabolických ciest vedie k ich akumulácii v krvi. Narušenie ureosyntézy spôsobuje vzostup amoniaku v krvi, ktorý sa v pokročilých štádiách ochorenia podieľa na rozvoji hepatálnej encefalopatie [8]. Esenciálne aminokyseliny s rozvetveným uhľovodíkovým reťazcom valín, leucín a izoleucín sú len nepatrne metabolizované v pečeni. Sú významným zdrojom energie pre svaly, ktoré ich aktívne vychytávajú Anesteziologie a intenzivní medicína 29 z krvi a môžu nahrádzať energiu pochádzajúcu z glukózy. Ich aminoskupiny tlmia acidózu tým, že sa premenia na NH4+ a sú vylučované obličkami. Podávanie spomínaných aminokyselín do výživy preto mohlo pozitívne vplývať aj na regeneráciu svalov a kompenzovanie acidózy, ktorá sa prejavila v danom prípade aj zvýšením laktátu v krvi. Podávanie gluta-mínu, ktorého syntéza v organizme vyžaduje ATP tiež mohlo významne pozitívne vplývať na regeneračné procesy, pretože glutamín je východiskovou látkou pri syntéze pyrimidínových a purínových báz, ktoré sú základnými bázami nukleových kyselín, ale aj energeticky bohatých triťosťátov ako ATP, GTP, UDP atď. Tie majú významnú úlohu okrem syntetických dejov i v regulácii metabolizmu prenosom signálov a usmerňovaním metabolizmu, napr. druhý poslovia (cAMP, cGMP). Prebytočný glutamín nevyužitý k syntéze spomenutých báz môže poskytnúť amoniak, ktorý pribratím H+ kompenzuje acidózu, podobne ako amoniak z rozvetvených aminokyselín. Selén sa uplatňuje ako kofaktor enzýmu glutatión peroxidázy, ktorý patrí do skupiny enzýmov vykazujúcich peroxidázovú aktivitu [10]. Jej hlavnou úlohou je ochrana bunkových štruktúr pred oxidačným poškodením. Biochemická funkcia GPx je redukovať lipido-vé peroxidy na im korešpondujúce alkoholy a redukovať voľný peroxid vodíka na vodu. Substrátom pre túto reakciu je glutatión (GSH) [11, 12]. Suplementá-ciou glutamínu alebo jeho prekurzorov sa zvyšuje efektivita účinku GPx. Regenerácia oxidovanej formy glutatiónu (GSSG) na GSH je katalyzovaná glutatión reduktázou (GR). Priame meranie dynamiky antioxi-dačných enzýmov u pacientky z kazuistiky poukazuje na ich aktiváciu suplementovanými mikronutrientmi. U pacientky z kazuistiky dochádza aj k zvýšeniu aktivity GR, čo je možné vysvetliť súčasným podávaním prekurzorov syntézy GSH. Zvýšená hodnota GPx v šiesty deň od aplikácie prvej dávky selénu a glutamínu svedčí o zvýšenej aktivite antioxidačného systému. Zabudovanie selénu do selenoproteínu, akým je GPx, a zvýšenie jej aktivity má význam v zabránení monoredukcie peroxidov na .OH, čo nie je enzymatická reakcia, ale môže byť značne urýchlená prítomnosťou kovov, napr. z porušených molekúl, kde sa ióny kovov často nachádzajú v ich aktívnom centre. Metalo-proteínom je aj superoxid dizmutáza (SOD), ktorá je ďalším dôležitým antioxidačným enzýmom. Jeho funkciou je rýchla dizmuťácia 02." na kyslík a peroxid vodíka. V prostetickej skupine tohto enzýmu sa nachádza med, zinok alebo horčík. Pred zahájením liečby bola aktivita tohto enzýmu znížená. Počas liečby došlo k zvýšeniu aktivity aj u tohto enzýmu. Biochemické parametre iných meťaloproteínov sledované neboli.Tento jav bol sprevádzaný poklesom plazmatickej hladiny CRP, čo je známkou efektívnej terapie systémovej zápalovej odpovede. Pomer neutrofilov a lymfocytov má priaznivú klesajúcu tendenciu aj napriek stúpajúcej hodnote leukocytov. Pomer neutrofilov a lymfocytov jednoducho a spoľahlivo vystihuje mieru závažnosti oxidačného stresu a systémového zápalu. Predošlé štúdie popisujú súvislosť medzi závažnosťou klinického stavu a mierou neutrofílie a lymfocytopénie [13]. Výsledky tejto kazuistiky potvrdzujú priaznivý vplyv su-plementácie selénu na SIRS a obnovu hepatálnej funkcie. Zvýšenie aktivít anťioxidačných enzýmov u pacientky z kazuistiky poukazuje na ich aktiváciu suplementovanými mikronutrientmi. Záver Stillova choroba je ochorenie neznámej etiológie, vyžadujúce dlhodobú imunosupresívnu liečbu. Akútna exacerbácia ochorenia môže viesť k rôznym orgánovým dysfunkciám a z nich vyplývajúcim klinickým prejavom. Jedným z najzávažnejších je akútna hepa-tálna insuficiencia, ktorá pri neadekvátnej terapii môže viesť k fulminantnému zlyhaniu pečene. Liečba je vysoko komplexná, vyžadujúca multidisciplinárny prístup. Pochopenie patofyziologických pochodov pri stavoch spojených so zlyhávaním pečene má kľúčovú úlohu pri výbere orgánovo špecifickej nutrície, ktorá adekvátne reaguje na metabolické potreby organizmu. Využitie podpornej antioxidačnej terapie formou selénových preparátov a prekurzorov glutatiónu sa zdá byť výhodné pri ovplyvnení systémovej zápalovej odpovede a zlepšení klinického stavu. Použité skratky: AS - anaplerotický nutričný substrát ATB - antibiotiká ATP - adenozíntrifosfát Ci-Ca - modul citrátový - kalciový modul cAMP - cyklický adenozínmonofosfát cGMP - cyklický guanozínmonofosát CVVHD - Continuous venovenous hemodiafiltration CRP - C-reaktívny protein DIC - disseminovaná intravaskulárna koagulopatia GCS - Glasgow coma scale GPx - glutatión peroxidáza GR - Glutatión reduktáza GSH - redukovaná forma glutatiónu GSSG - oxidovaná forma glutatiónu GTP - guanozíntrifosfát MEL - mimotelová eliminačná liečba 02•" - superoxid .OH - hydroxylový radikál PCT - prokalcitonín ROS - reaktívne formy kyslíka SIRS - Systemic Inflammatory Response Syndrome SOD - superoxid dismutáza ŠS - špecifický nutričný substrát UDP - uridíntrifosfát UVP - umelá ventilácia pľúc Anesteziologie a intenzivní medicína 341 30 Literatura 1. Bywaters, E. G. L. Still's disease in the adult. Ann. Rheum. Dis., 1971, 30, p. 121-132. 2. Keher, J. P. Free radicals as mediators of tissue injury and disease. Critical rev. Toxicol., 1993, 23, p. 21-48. 3. Geoghegan. M., Mcauley, D., Eaton, S., Powel-Tuck, J. Selenium in critical illness. Critical Care, 2006, 12, p. 136-141. 4. Kočan, L., Firment, J., Šimonové, J., Vašková, J., Guzy, J. Suplementácia selenu u pacientov s fažkou akútnou pan-kreatitídou. Rozhl. v Chirurgií, 2010, 89,1, p. 518-521. 5. Gartner, R., Albrich, W., Angustwurm, M. W. The effect of selenium supplementation on the outcome of patients with severe systemic inflammation, burn and trauma. Biofactors, 2001, 14, p. 199-204. 6. Imlay, J. Pathways of oxidative damage. Annual Review of Microbiology, 2003, 57, p. 395-418. 7. Andrews, P. J. Selenium and glutamine supplements: where are we heading? A critical care perspective. Current Opinion in Clinical Nutrition and Metabolic Care, 2010, 13, p. 192-197. 8. Zadák, Z. Umělá výživa při poškození jater. In Zadák, Z. Výživa v intenzivní péči. Grada Publishing a.s, : Praha, 2008, p. 359-365, ISBN: 978-80-247-2844-5. 9. Turecký, L. Poškodenie pečené volnými radikálmi. In Ďurač- ková, Z. Voľné radikály a antioxidanty v medicíne II. Slovac Academie Press s.r.o. 1999, p. 210-218.ISBN: 80-88908-46-9. 10. Berger, M., Chiolero, R. Antioxidant supplementation in sepsis and systematic inflammatory response syndrome. Critical Care Medicine, 2007, 35 p. 584-590. 11. Forceville, X. Seleno-enzymes and seleno-compounds: the two faces of selenium. Critical Care, 2006,10, p. 180. 12. Muller, F. L., Lustgarten, M. S., Jang, Y., Richardson, A., Van Remmen, H. Trends in oxidative aging theories. Free Radio. Biol. Med., 2007, 43, p. 477-503. 13. Záhorec, R. Pomer neutrofilov a lymfocytov - rýchly a jednoduchý ukazovateľ systémového zápalu a stresu u pacientov v kritických stavoch. Bratislavské Lekárske Listy, 2001, 102, p. 5-14. Došlo dne 11.8.2011. Přijato dne 11. 11.2011. Adresa pro koresponddenci: MUDr. Ladislav Kočan I. klinika anestéziológie a intenzívnej medicíny Univerzitná nemocnica L. Pasteura Trieda SNP 1 041 90 Košice Slovenská republika e-mail: kocanladislav@yahoo.com 342 Anesteziologie a intenzivní medicína 31 2.5.3 Závery štúdie SE-AOX Deficit selénu u kriticky chorých pacientov sme potvrdili aj našimi meraniami prostredníctvom atómovej absorbčnej spektrofotometrie. Výsledky týchto meraní boli prezentované na postgraduálnom kurze Sepsy a MODS v Ostrave 2010. • Doterajšie skúsenosti liečby Selénom u kriticky chorých / J. Firment, L. Kočan, J. Kubálková. [Up to now experiences in selenium treatment in criticaly illj. In: 12. postgraduální kurz Sepse a MODS : 26.-29. leden 2010. - Ostrava, 2010. - ISBN 9788025480397. - S. [1-3]. Vyhodnotením výsledkov štúdie Se-AOX neboli zistené rozdiely v mortalite, výskyte renálneho zlyhania a respiračných funkcií medzi jednotlivými skupinami a podskupinami (p>0,05). V obidvoch skupinách, selén versus placebo, bolo preukázané, že pacienti s vyšším skóre APACHE II a SOFA mali preukázateľne vyššiu úmrtnosť (p<0,05). Medzi skupinami a podskupinami neboli zistené významné rozdiely dynamických zmien sledovaných biochemických parametrov (CRP, laktát, celkové bielkoviny, albumín, urea) (p>0,05), hematologických a koagulačných parametrov (počet leukocytov a ich populácii, trombocytov, fibrinogénu) (p>0,05) počas terapie. Zmeny aktivít antioxidačných enzýmov v sledovaných podskupinách potvrdili nárast aktivít GPx u pacientov, ktorým bol suplementovaný Se (p<0,05). Pacientom v podskupine 01 < 200, ktorým sa suplementoval selén, sme zaznamenali signifikantné zlepšenie oxygenačného indexu na konci terapie selénom (p<0,05), ale bez ďalšieho klinicky významnej korelácie. Výsledky štúdie nepreukázali výrazný klinický benefit u pacientov, ktorým sa suplementoval selén oproti placebo-skupine, tento rozdiel nebol zaznamenaný ani medzi jednotlivými podskupinami. Zároveň výsledky štúdie Se-AOX korelovali s výsledkami ďalších, v tom čase aktuálnych prospektívnych randomizovaných klinických štúdií, akými bola štúdia SIGNET Andrews a kol., 2011 (4), ako aj štúdie Valenta a kol., 2011 (3). Na základe v tej dobe zistených výsledkov boli doplnené odporúčania pre liečbu sepsy mikroprvkami zhrnuté v Surviving Sepsis Campaign: International Guidelines for Management of Sepsis and Septic Shock: 2016. Intensive Care Medicíne, v ktorých sa podávanie selénu pri liečbe sepsy a septického šoku ďalej neodporúča (silné odporúčanie, stredná kvalita dôkazov) (21). Závery štúdie Se-AOX boli publikované vo: Wiener Klinische Wochenschrift - ISSN 0043-5325, IF 1,3, Q2 a Clinical Biochemistry - ISSN 0009-9120, IF 2,3, Q2. 32 original article Wien Klin Wochenschr (2013) 125:316-325 DOI 10.1007/S00508-013-0371-x Wiener klinische Wochenschrift The Central European Journal of Medicine Restoration of antioxidant enzymes in the therapeutic use of selenium in septic patients Vašková Janka, Kočan Ladislav, Firment Jozef, Vaško Ladislav Received: 3 December 2012 / Accepted: 10 April 2013 / Published online: 4 May 2013 © Springer-Verlag Wien 2013 Summary A prospective observational study of parenteral selenium supplementation started in January 2008 which included 72 septic patients with APACHE II scores ranging from 19 to 40 after admission. Patients were divided into two major groups: one with a continual infusion of sodium selenite at 750 ug/24 h for 6 days and a placebo group followed by subgroups according to the presence or absence of surgical procedure. Routine biochemical and hematological parameters were determined continuously. Sequential Organ Failure Assessment (SOFA) scores were calculated in two-day intervals. Patients who died had a higher Acute Physiology and Chronic Health Evaluation (APACHE) II score, lower albumin on the 3rd days of therapy and higher C-reactive protein (CRP) on the 6th days of therapy. Statistically, there was no significant difference in the comparison of CRP, fibrinogen, albumin, plasma proteins, or neutrophil to lymphocyte counts during the 6 days in all subgroups. There was a significant difference in the comparison of leukocytes on the 6th day of therapy. Glutathione peroxidase and glutathione reductase activity was increased in selenium subgroups with negative correlation in placebo subgroups during the therapy. A downward trend in SOD activity, more appreciable in selenium groups, seemed to be a reflection of lower superoxide radical production. This is biased more as a result of GPx activity restoration, preventing further peroxidation of or- L. Kočan, PhD (El) • Assoc. Prof. J. Firment, PhD 1st Clinic of Anaesthesiology and Intensive Care Medicine, Louis Pasteur University Hospital in Košice, Tr. SNP 1, 04066 Košice, Slovak Republic e-mail: kocanladislav@yahoo.com J. Vašková, PhD • Assoc. Prof. L. Vaško, PhD Institute of Medical and Clinical Biochemistry, Faculty of Medicine, Pavol Jozef Šafárik University in Košice, Košice, Slovak Republic ganic substrates and cyclic formation of other radicals, than actual attenuation of their production. Selenium substitution increased selenium dependent antioxidant enzyme activity and, in comparing mortality in groups, we found a 16.7 % decrease in mortality in favor of supplementation with selenium. Keywords: Oxidative stress, Sepsis, Selenium, Artificial nutrition Wiederherstellung der Aktivität von antioxidativen Enzymen durch Selen bei septischen Patienten Zusammenfassung Eine prospektive Beobachtungsstudie der Wirkung einer parenteralen Gabe von Selen wurde im Jänner 2008 begonnen. Es wurden 72 Patienten mit Sepsis und einem APACHE II Score zwischen 19 und 40 bei Aufnahme in die Studie aufgenommen. Die Patienten wurden in 2 große Gruppen eingeteilt: einer Gruppe wurde eine kontinuierliche Infusion von 750 |ig Na-Selenit/24 h 6 Tage lang verabreicht - der anderen Placebo. Es wurden dann Subgruppen gebildet, je nachdem, ob operative Maßnahmen gesetzt wurden. Routine biochemische und hämatologische Parameter wurden kontinuierlich erhoben. Die SOFA Scores wurden alle 2 lägen errechnet. Die verstorbenen Patienten hatten einen höheren APACHE II Score, sowie ein niedrigeres Albumin am 3. Tag und ein höheres CRP am 6. Tag. Statistisch bestand kein signifikanter Unterschied im Vergleich des CRP, des Fibrinogens, des Albumins, der Plasmaproteine bzw. der Neutrophilen und Lymphozyten Zahl aller Subgrup-pen während der 6 Beobachtungstage. Die Leukozyten-zahl war am 6. Tag statistisch signifikant unterschied-lich. Die Aktivität der Glutathionperoxidase (GPx) und der Glutathionreduktase war in den Selen-Subgruppen 316 Restoration of antioxidant enzymes in the therapeutic use of selenium in septic patients Springer 33 original article während der Therapie erhöht mit einer negativen Korrelation in den Placebo-Untergruppen. Ein Abwärtstrend der SOD Aktivität, der in den Selen-Untergruppen deutlicher war, schien eine Folge einer geringeren Produktion von Superoxyd Radikalen zu sein. Wahrscheinlich ist das aber eher auf eine Wiederherstellung der GPx Aktivität (die vor einer weiteren Peroxydation der organischen Substrate und einer zyklischen Bildung von anderen Radikalen schützt) als auf eine tatsächliche Verminderung ihrer Produktion zurückzuführen. Substitution mit Selen erhöhte die Aktivität der Selenabhängigen antioxidativen Enzyme. Ein Vergleich der Mortalität beider Gruppen ergab eine um 16,7% niedrigere Sterberate der mit Selen behandelten Patienten. Schlüsselwörter: Oxidativer Stress, Sepsis, Selen, Künstliche Ernährung Introduction Many serious medical problems originate from conditions of oxidative stress, that are amplified in the immune response. Its activation role in the pathogenesis of syndromes such as systemic inflammatory response syndrome (SIRS), sepsis, severe sepsis, septic shock, and in various disease states such as acute pancreatitis is well known, and also in other diseases encountered in the intensive care unit (ICU) [16]. Treatment of such diseases is very complex, involving causal treatment, support, and assistance. An individual approach is used from one patient to another. Oxidative stress can be influenced by choosing the optimal enteral and parenteral nutrition, which can, among other things, activate the body's antioxidant defense and add missing trace elements and vitamins which have been consumed. The current multicenter study confirmed the importance of supplementation of these nutrients to support antioxidant capacity of the organism. Decreased plasma levels of antioxidants are caused by increased antioxidant consumption in plasma and tissues, necessary for the destruction of free radicals. The decrease is considerable in uric acid, protein and thiol groups, unconjugated bilirubin, ascorbic acid, alpha-tocopherol, beta-carotene, lycopene, antioxidant enzymes, glutathione, and trace elements such as selenium and zinc [7]. Absorption of dietary selenium in the inorganic form is limited to about 50%, while the organic form is readily taken up, with up to 100 % of organic dietary selenium being absorbed. After resorption in the small intestine, it is transported in the blood bond to selenoprotein P and its plasma level is sensitive to psychological and physical stress. Also selenoprotein P represents a stock potential in which over 50 % of plasma selenium is stored. The level of this protein is a determinant of the total selenium status [28], which is, however, insufficient in ac ute co nditions. The safe oral intake for healthy people with normal renal function is 50-200 ug/day [15]. Selenium balance in the body is regulated by the kidneys. Adults excrete 50-60 % of selenium in the urine. Selenium in the body forms part of some proteins, most of which play an important role in protecting cell structures from oxidative damage. The most important macromolecule of this group is an enzyme, glutathione peroxidase [28], although selenium is also found in the active sites of two other important enzymes in the metabolism of thyroid hormones: jodotyronine-5-deio-dinase, and that of nucleotides, thioredoxin reductase. Growing clinical studies in patients with SIRS, sepsis, burns, trauma, Acute respiratory distress syndrome (ARDS), and pancreatitis suggest that selenium supplementation may be advantageous. Administration of selenium-containing supplements appears to be beneficial in terms of reducing mortality [13]. Ingestion of 4 mg of sodium selenite pentahydrate is considered nontoxic in healthy humans [12]. Critically ill patients have lower levels of selenium in their blood compared with the healthy population, so are much more able to tolerate the increased supply of selenium without showing signs of toxicity [2]. An intake of 1,000 ug/day of sodium selenite pentahydrate administered intravenously was tolerated well by patients [13]. Several clinical studies have been devoted to the issue of selenium antioxidant therapy in selected pathologies [2, 12, 15, 18]. Individual studies addressing this issue are varied in respect to the dose and duration of selenium administration, the form of administration (bolus vs. continuous administration) and controversial results. The reality clarified for us the relationship between the dose and the clinical response. On this basis, we developed a methodology of the study named Se-AOX in early 2008. From previous studies and meta-analyses, it has been concluded that a safe interval of administration of sodium selenite pentahydrate falls in the range of 600-800 ug per day. However, other studies were also published later in which high doses of sodium selenite pentahydrate were administered on the first day at 1,000 ug/day and from days 2 to 14 at 1,500 ug/day in an attempt to exploit the prooxidant properties of selenium in the initial stage of oxidative stress on neu-tralization of microbial pathogens and the next phase of its antioxidant properties after installation into proteins. The results of the study demonstrated no statistically sig-nificant reduction in mortality in any groups [1, 29]. In our prospective observational study, Se-AOX, we studied the dynamic changes of antioxidant enzymes, the development of biochemical and hematological parameters of sepsis, and 28-day mortality in critically ill patients with APACHE II (median 24, min 19, max 40) and SOFA (median 9, min 4, max 14), treated with sodium selenite pentahydrate at a dose of 750 ug/day as a continuous infusion for 6 days. Patients, materials and methods The 1st Clinic of Anaesthesiology and Intensive Care Medicine, University Hospital of Louis Pasteur in Košice, Slovak Republic started a prospective observational study, Se-AOX, in 2008. The study looked at the effects of 4y Springer Restoration of antioxidant enzymes in the therapeutic use of selenium in septic patients 317 34 original article parenteral selenium supplementation in septic patients and subsequent monitoring of dynamic changes in the activities of selected antioxidant enzymes and the development of clinical status. A protocol has been developed, on the basis of which the University ethics committee approved the study 109/2011. As the study was observational, therapeutic procedures were performed in our clinic routinely and most of the patients had altered consciousness, the patients' special informed consent was not requested. The Se-AOX study enrolled 72 patients with an APACHE II score between 19 and 24 points at the ages from 23 to 79 years, who developed sepsis, severe sepsis or septic shock during hospitalization as defined by the International Sepsis Definitions Conference [ 19]. A SOFA score was calculated in the time periods of 1-2 days (Tl), 3-4 days (T2) and 5-6 days (T3) according to an internet calculator (http:// www.sfar.org/scores2/sofa2.php). The Se-group consisted of 35 patients who received selenium supplementation during hospitalization in the form of sodium selenite pentahydrate at 750 ug/day for 6 days immediately after admission to our department (1,000 ug of sodium selenite pentahydrate =333 pg of selenium) (Selenase, Vivax; selenium hereinafter). The placebo group consisted of 37 patients who received con-tinuous saline NaCl 50ml/day for 6 days as a continuous infusion (excluding additional infusion therapy). Patients were further divided into four subgroups according to whether they received surgery. The Al subgroup con-sisted of 23 patients who received selenium supplements postoperatively. Subgroup A2 consisted of 21 patients treated with placebo during the postoperative period. The Bl subgroup consisted of 12 patients who received selenium supplements without undergoing surgery. Finally, the B2 group consisted of 16 patients treated with placebo without undergoing surgery (Fig. 1). The nutritional strategy (enteral vs. parenteral, qualitative and quantitative characteristics of the diet) was dependent on the patient's diagnosis and nutritional sta-tus, as well as the standard therapeutic approaches to the disease. Food administration was cyclical, to stimulate the diurnal rhythm. The daily dosage of glucose sugar was from 1.5 g/kg up to 5.5 g/kg. The total daily supply of amino acids ranged from 0.8 to 2.5 g/kg (0.5-0.75 g/kg for renal and liver injury, 0.7-1.0 g/kg in stable patients, 1.0-1.5 g/kg in a poor state of nutrition and catabolism, and 1.5-2.0 g/kg for severe malnutrition). Substitution of fat was performed after the acute stage of the dis-ease subsided and was administered at 0.5 g/kg/day. All groups were also administered antioxidants paren-terally: 1,000 mg/24 h vitamin C, 30 mg/24 h vitamin E, 100 mg/24 h thiamine, 100 mg/24 h pyridoxine, and 1,000 u.g/24 h cobalamin. The determination of biochemical and hematological parameters of blood samples formed part of the routine diagnostic methods. Data collection for the Se-AOX study was performed at two-day inter-vals: Tl (lst-2nd day), T2 (3rd-4th day) and T3 (5-6th day). Kinetic methods for estimating the activities of glutathione peroxidase (GPx, E.C. 1.11.1.9) and glutathione reductase (GR, E.C.1.6.4.2) were performed using a kit (Sigma-Aldrich, Germany) and that of superoxide dismutase (SOD, E.C. 1.15.1.1) by means of the SOD-Assay Kit-WST (Fluka, lapan) following the user manual provided. A control group of 30 healthy volunteers was used for comparison with the development activity of antioxidant enzymes in the critically ill. Statistical significance was determined by T-test (calculated from mean, SD) baseline vs. 6-7th day (T3) and Pearson's Chi-square selenium vs. non-selenium group from baseline to 6-7th day. Differences were considered significant at p<0.05. Possible benefit from the treatment was calculated by number needed to treat (NNT) test. Results There was a decrease in the median SOFA scores of surgical patients, in both selenium and placebo groups (Al and A2), and nonsurgical patients (Bl) at time periods Tl, T2, and T3 (Table 1). This suggested a reduction in the number of organ failures and an improvement in health. There was a decrease in median CRP levels in all groups followed (Table 2). There were no significant differences (p>0.05) between the observed biochemical parameters shown in Table 2. In group Al—surgical patients receiving selenium supplements—median platelet values were increased (Table 3), suggesting both the incorporation of selenium and the mitigation of the systemic inflammatory response. Through the course of treatment, there was a decreasing tendency for the median values of white blood cells in both groups of nonsurgical patients (Bl and B2) which translates to a significant change in values between groups A and B at T3. Patients in both the A subgroups had a decreased 28-day mortality of 16.7 % in comparison to group B (Table 4). The reduction was not statistically significant (p> 0.05), although the figure is not at all negligible. Using NNT, we can state with 95 % certainty that the absolute reduction in 28-day mortality was 16.6 % and that every 7th patient benefits from the treatment. High SOD activity values were observed during this period in surgical patients (Table 5). The median of the measured values has a slightly decreasing trend in groups Al and A2; however, unlike in groups without selenium administration, these values returned toward the median of healthy volunteers. The activity of GPx was increased in the interval T1-T3 in patients supplemented with selenium from groups Al and Bl, but was pronounced in the group in surgical patients, Al. It is in these patients that the median values were closest to the median values of healthy volunteers, albeit without reaching them. Similarly, an upward trend could be observed in group Bl. A much weaker upward trend could be observed in the group with surgery without selenium supplementation (A2), and in group B2 (significance atP<0.05 in comparison to Bl). Median values of GR in Al are very close to the median values of healthy volunteers, although there is a slight downward trend when compared with baseline 318 Restoration of antioxidant enzymes in the therapeutic use of selenium in septic patients ^ Springer 35 original article Critically ill patients with signs of sepsis, severe sepsis, septic shock APACHE II 19-60 (n=95) Loss to follow up (n= 15) Patients with APACHE <19, >40 excluded (n=80) Patients met inclusion criteria APACHE 19-40 Lost to follow-up (died in day after admission) (n=8) 1(n=35) Started selenium adjuvant therapy immediately after the diagnosis of sepsis, decisive criterion was even date of admission to ICU 2(n=37) Without selenium adjuvant therapy, decisive criterion was odd date of admission to ICU Al (n=23) Patients within 24 hours after admission underwent surgery A2 (n=21) Patients within 24 hours after admission underwent surgery Bl (n=12) Patients did not undergo surgery B2 (n=16) Patients did not undergo surgery Fig. 1 Flow diagram of prospective observational study Se-AOX in septic patients in groups with selenium adjuvant therapy l and placebo 2, with surgical (A) and without surgical performance (B) values during T2-T3 (only for median values). The case with A2 is similar. The differences in the changes in GR activity are generally available for monitoring when comparing groups A and B. The median B values are lower, although the differences between the groups are not statistically significant. This may be a result not only of fewer patients, as they were divided into four groups, but also of the range of values measured and thus higher deviations from the mean. Discussion Normally, there is homeostasis between the production of reactive oxygen radicals and their removal by endogenous antioxidant scavengers, nonenzymatic antioxidant supplementation, and diet. Oxidative stress results from an impaired balance of excessive reactive oxygen species (ROS) production, including superoxide, hydrogen peroxide and hydroxyl radicals and/or inadequate antioxidant defense. Severe infections and inflammatory Springer Restoration of antioxidant enzymes in the therapeutic use of selenium in septic patients 319 36 original article Table 1. Demographic characteristics of the patients in groups with selenium adjuvant therapy A1-with surgical and F31-without surgical performance and placebo groups A2-with surgical and B2-without surgical performance Characteristics Median (min-max) Median (min-max) Ai A2 B1 B2 N 23 21 12 16 Age (23-78) 53 (30-77)60 (28-68) 47 (40-79) 53 Gender (M/F) 17/6 14/7 6/6 12/4 APACHE score (19-39) 24 (19-38)23 (20-40) 24 (19-37)30 SOFA score T1 (4-14)10 (6-13)9 (3-14)8 (4-14) 9 SOFA score T2 (1- 14)9 (4-17)8 (1-14)8 (2-12)9 SOFA score T3 (1- 15)8 (2-19)6 (2-14)6 (3-14)9 Sepsis 4 5 3 5 Severe sepsis 5 8 5 4 Septic shock 14 8 4 7 Disseminated intravascular coagulation 4 1 1 2 Bronchopneumonia 8 7 6 8 ALI 8 11 2 3 ARDS 11 1 5 6 Nosocomial infections 11 6 4 9 Dialysis 13 6 5 5 Admission diagnosis Respiratory failure 21 12 7 14 Coma (status after CPR) - 2 2 3 Cardiac disease 1 - 1 3 Hemato-oncological disease - - 2 4 Peritonitis 8 7 - - Pancreatitis 10 2 - - State after neurosurgical procedure 2 - - - State after cardiosurgical procedure 1 1 - - State after urological procedure 1 3 - - Trauma 2 1 - - Hemorrhagic shock 3 2 - - Table 2. Comparison of the levels of selected parameters in plasma over T1-T3 day interval Parameter (ref- Time Median (min-max) P Median (min-max) P P(Avs. B) erence values) interval ^ A2 B1 B2 Total plasma T1 49.0 (36.0-62.2) 49.0 (39.8-63.4) 0.09 50.5 (36.0-77.9) 46.8 (27.4-78.5) 0.90 0.22 protein T2 50.6 (34.0-62.5) (64.0-83.0 g/l) T3 53.6 (36.3-60.3) 51.2(42.2-70.0) 50.0 (34.1-65.0) 0.96 0.23 50.0 (31.5-68.0) 50.1 (31.5-73.4) 49.9 (28.1 -73.8) 49.8 (34.6-70.4) 0.40 0.99 0.56 0.37 Albumin T1 22.8(13.4-39.0) 25.3 (20.2-37.9) 0.23 25.3 (14.5-50.0) 25.6 (34.6^13.7) 0.77 0.19 (35.0-52.0 g/l) T2 1 g7 (12.3-32.3) T3 21.8(12.9-39.2) 27.0(21.6-32.1) 0.09 22.9 (14.9-39.4) 20.7 (14.3-37.2) 0.17 0.45 26.4(17.8-35.3) 0.56 24.5 (12.5-39.2) 23.1 (13.4-35.6) 0.48 0.89 CRP T1 180.4(38.2-482 0) 148.8 (23.6-367.2) 0.12 103.4 (4.5-308.8) 156.3 (15.6-445.3) 0.60 0.22 (0.1-5.0 mg/l) T2 190.2(27.4-356 6) 97.0 (12.2-428.5) 0.20 93.5(12.2-444.5) 108.5(51.6-386.5) 0.61 0.47 T3 168.7(20.7-482 1) 98.3 (2.7-242.8) 0.07 115.5 (10.2-318.0) 95.0 (36.1 -249.0) 0.31 0.21 Fibrinogen T1 5.4 (2.8-8.7) 4.4 (0.8-8.4) 0.25 4.6(1.9-8.4) 4.2(2.0-8.9) 0.79 0.51 (1.8-3.5 g/l) T2 5.7(1.8-8.9) 4.9(1.2-7.0) 0.22 4.4(1.1-9.0) 5.1(3.0-14.0) 0.25 0.92 T3 5.3(1.7-10.9) 5.0 (2.5-8.5) 0.16 5.0(1.3-7.8) 5.0(2.7-12.0) 0.42 0.58 320 Restoration of antioxidant enzymes in the therapeutic use of selenium in septic patients Springer 37 original article Comparison of the number of blood components and ratios of neutrophils/lymphocytes in subgroups Parameter (ref- Time Median (min-max) P Median (min-max) P P(Avs.B) erence values) interval A1 A2 B1 B2 Platelets (150- T1 152.5 (21.7-499.3) 233.0 (41.7-410.0) 0.64 204.0 (6.6-550.0) 121.3 (30.7-326.5) 0.10 0.53 350x10s/!) T2 192.2(30.7-432.3) 198.0 (27.7-322.0) 0.99 132.0 (49.0-445.0) 104.0 (12.5-414.0) 0.70 0.76 T3 221.5(48.0-483.0) 153.0(13.0-337.0) 0.32 123.0 (57.5-477.0) 84.5 (16.5-526.0) 0.92 0.41 WBC (4.0- T1 14.8(5.5-32.2) 13.4(3.8-21.4) 0.56 15.7(3.1-30.7) 10.8 (1.9-62.2) 0.16 0.96 10.0x109/l) T2 12.6(3.1-31.3) 10.3 (5.5-30.9) 0.38 12.3(4.1-29.3) 12.8(2.1-34.9) 0.05 0.96 T3 13.4(1.5-25.0) 14.2(6.5-27.4) 0.48 9.9(3.5-18.6) 8.5 (1.0-29.3) 0.09 0.003* Neutrophils T1 15.33(2.95-32.47) 12.1 (3.2-18.4) 0.32 11.0(6.0-28.1) 9.4 (0.9-24.8) 0.32 0.11 (33-80 %) T2 11.25(2.59-29.60) 8.0(5.0-16.4) 0.86 11.7(5.6-24.9) 9.0(0.9-19.4) 0.84 0.93 T3 13.40(1.30-17.80) 6.8 (1.7-3.7) 0.78 11.2 (1.1-20.1) 8.1 (4.6-24.7) 0.47 0.74 Lymphocytes T1 1.02(0.33-8.24) 1.1 (0.5-2.5) 0.28 1.1 (0.7-1.8) 0.9 (0.1-2.6) 0.49 0.16 (15-35%) T2 1.11 (0.31-8.19) 1.1 (0.2-2.2) 0.73 1.2(0.6-2.3) 0.8 (0.1-2.3) 0.66 0.59 T3 0.75(0.14-9.10) 1.8(0.2-2.4) 0.92 1.1 (0.1-2.0) 1.1 (0.3-2.3) 0.54 0.78 Neu/Ly ratio T1 5.38 (2.80-8.71) 4.4 (0.8-8.4) 0.43 4.6(1.9-8.4) 4.2 (2.0-8.9) 0.50 0.18 (0.94-5.33) T2 5.73 (1.79-8.93) 4.9 (1.2-7.0) 0.25 4.4 (1.2-9.0) 5.1 (3.0-14.0) 0.90 0.12 T3 5.32 (1.73-10.85) 5.0 (2.5-8.5) 0.95 5.0 (1.3-7.8) 5.0(2.7-12.0) 0.42 0.23 *p<0.01 Table 4. Mortality of patients in the groups dosed with selenium and without selenium administration (x2 = 1.985, p= = 0.159) Group of patients Se-adjuvant therapy Mortality Total Survived Died 1 + 20 15 35 2 - 15 22 37 Total 35 37 72 Table 5. Dynamic changes in the activities of SOD, GPx, and 3R in subgroups A and B at time intervals T1, T2, and T3 Parameter (healthy Time Median (min-max) P Median (min-max) P P(Avs.B) volunteers-median interval ^ A2 B1 B2 (min-max) SOD 3.11 T1 4.83 (3.65-8.89) 4.89 (3.80-8.81) 0.89 3.96 (3.44-6.95) 4.01 (3.44-7.69) 0.99 0.35 (2.44-5.21) (U/ml) T2 4.36 (3.38-6.03) 3.89 (3.28-5.74) 0.54 4.05 (3.26-5.55) 4.11 (2.98-6.01) 0.42 0.30 T3 4.25(3.15-6.19) 3.69 (3.05-5.13) 0.19 3.95 (3.25-6.05) 3.88 (3.56-5.23) 0.99 0.07 GPx 1.12 (0.858- T1 0.177 (0.017-1.002) 0.182 (0.017-0.995) 0.97 0.215(0.050-0.80) 0.123 (0.064-0.760) 0.96 0.73 6.066) (ukat/l) T2 0.333 (0.011-1.480) 0.155 (0.048-0.367) 0.50 0.325 (0.117-1.317) 0.200(0.012-1.766) 0.14 0.85 T3 0.833 (0.105-1.650) 0.194 (0.093-0.267) 0.16 0.692(0.067-1.233) 0.300(0.019-1.017) 0.04* 0.28 GR 1.15 (0.567- T1 1.084 (0.033-1.278) 1.076 (0.034-1.219) 0.98 0.283(0.033-1.770) 0.283 (0.033-1.770) 0.94 0.06 3.917) (ukat/l) T2 0.801 (0.117-4.947) 0.486(0.133-3.383) 0.44 0.367(0.017-1.932) 0.660(0.050-1.113) 0.17 0.63 T3 0.852 (0.050-4.600) 0.580 (0.033-2.217) 0.41 0.458(0.017-3.083) 0.712 (0.083-1.602) 0.11 0.80 *p<0.05 conditions in sepsis, septic shock, and SIRS cause periods of strong oxidative stress, which is characterized by a dramatic decrease in the levels of antioxidants and their cofactors, leading to the development of severe complications such as ARDS and multiple organ failure (MOF). Septic patients have a significantly reduced level of plasma selenium with antioxidant, anti-inflammatory, and immunological functions [14, 26], which is directly related to their high mortality rates. Although low levels of selenium in the plasma do not necessarily indicate a lack of selenium, it is reflected in selenoprotein activity, particularly in reduced GPx activity, thereby weakening the antioxidant enzyme system, or systems, for which the activity is necessary. This suggests that selenium deficiency in the critically ill is one of the causes of failure to cope with conditions of oxidative stress [2, 25]. Following the selenium status is difficult because of the redistribution of selenoproteins like albumin in septic patients due to increased permeability of blood vessels into the tissues, where it is preferentially incor- Springer Restoration of antioxidant enzymes in the therapeutic use of selenium in septic patients 321 38 original article porated into various selenoproteins [29]. The primary effect of sodium selenite is its prooxidant character, and its function as an antioxidant bound into selenoenzymes. Therefore, we have primarily chosen to follow changes in the activity of the antioxidant enzyme GPx. Since GPx belongs to a group of eight enzymes exhibiting substrate-specific peroxidase activity, which may be dependent or independent of selenium, the properly chosen methodology made it possible to measure the serum selenoen-zyme activities which eliminate peroxides and selenones faster than thiols. During these reactions, two electrons are transferred from reduced glutathione to peroxide which then decomposes to water or the corresponding alcohol. Patients who have been administered with selenium supplementation had a gradual increase in the GPx enzyme activity measured at time intervals Tl, T2, and T3 (Table 5). Group Al surgical patients had lower median values of GPx activity compared with patients in nonsurgical group Bl (Table 5), which may reflect the higher stress load to that group. GPx enzyme activity values in the Al subgroup at the time interval T3 (5-6th day) are higher than those in subgroup Bl. This may indicate higher antioxidant defense activity in surgical patients. Comparing surgical and nonsurgical subgroups, patients had comparable median values of GPx activity at time Tl. During therapy, a significant increase in GPx activity occurred in the Al subgroup not only compared to subgroup A2, but also compared to nonsurgical patients. A significant increase in activation occurred in patients receiving selenium supplements in Al vs. B2 (p<0.05), although the last day revealed that levels still remained below average compared with healthy subjects. The results, in agreement with previous studies as discussed by Mishra et al. [25] suggest a restoration of selenoen-zyme activities due to trace element supplementation. However, it is known that plasma GPx is an easily renewable selenoprotein, while other components of blood containing GPx like erythrocytes and platelets are still required to restore long-term supplementation [10]. According to Manzanaresetal. [21] andValentaetal. [29], GPx activity increases from the first day of Se supplementation and peaks at day 7, before falling on the 10th day regardless of continued supplementation. For this reason, we refrained from monitoring enzymatic activities further. A possible explanation for this phenomenon is considered to be enzyme inhibition or saturation due to lack of precursor synthesis, such as selenium hydride or selenocysteine, or limited synthesis of glutathione due to glutamine or cysteine deficiency [22]. Patients included in the study were given an enteral or parenteral form of alanylglutamine and other glutathione precursors to enhance protein synthesis. SOD activity has been identified as a suitable parameter for assessing oxidative stress in septic patients. Increased SOD activity was detected in the plasma and in various organs, e.g., heart, liver, and kidneys with increased formation of OJ. In addition, plasma SOD activity has been identified as predictive for patient sur- vival for approximately 3 h after the development of sepsis [27]. Higher SOD activities were observed in patients with septic shock [17]. The patients who have been reported as having low levels of SOD had a higher mortality [3]. However, little is known as yet about the activity of this enzyme in relation to selenium supplementation. There were significant differences in the treatment groups between the median values of input SOD activities of surgical and nonsurgical patients (Table 5). This phenomenon may be related to higher production of 02 in surgical patients, which requires more powerful elimination. During therapy, there was only a slight decrease in SOD activity in all four subgroups. Some authors believe, and we identify with this statement, as we will explain later, that the increase in SOD activity is associated with activation of neutrophils and macrophages, which leads to the production of Oj in defending the body against microorganisms, as these are dismutated by the catalytic effectiveness of SOD. The increase in SOD activity may also occur due to the increased production of free radicals by lipid peroxidation. Increasing SOD activity, however, is an effective antioxidant defense [8]. Under normal circumstances, Oj is formed by monovalent reduction of molecular oxygen in every living organism, and especially within the mitochondrial electron transport. Dismutation of 02 to hydrogen peroxide through SOD activity without concomitant rapid degradation of resulting peroxide by catalase and GPx may lead to the formation of a reactive hydroxyl radical. SOD activity values at Tl are higher than in healthy subjects, indicating that there is SOD activity without selenoenzyme support, but prooxidant in terms of the reduced effective defenses against emerging peroxide. Antioxidant defense support by selenium and glutathione precursor supplementation act in the sequence of reactions beyond those catalyzed by SOD. We can assume, therefore, that an increase in peroxide formation occurs as a result of increased SOD activity, whose excess generally causes inhibition of peroxidases and can count as another reason for the low GPx activity. The downward trend of SOD activity, more appreciable in A groups, seems to be a reflection of lower Oj production in time intervals T2 and T3 in all four subgroups (A vs. B not quite statistically significant at T3). We cannot draw a conclusion on the reduction of endogenously formed GPx, preventing further peroxidation of organic substrates and cyclic formation of other radicals, due to slight changes in the environment, such as changes in the number of neutrophils (Table 3). Another important enzyme involved in oxidation-reduction reactions in the elimination of free radicals is GR, which regenerates oxidized glutathione. The group of surgical patients in the initial measurement had higher GR activities than the nonsurgical group. Increased GR activity suggests an increased need for reduction of oxidized glutathione and indirectly points to intense oxidative stress in surgical patients. Subset A had an irregular downward trend following dynamic development of GR activity during T2-T3, as in a subgroup Bl. Measuring the Springer 322 Restoration of antioxidant enzymes in the therapeutic use of selenium in septic patients 39 original article GR activity in non-selenium subsets B2 confirmed only a small gradual rise in the median values (Table 5). On the one hand, we think that the generally low GR activity in critically ill patients compared to healthy subjects may be due to GSH level depletion. As already mentioned, there is limited synthesis of glutathione owing to glutamine or cysteine deficiency [22], Supplementation of glutathione synthesis precursors was used in an attempt to counteract this deficiency. Given that its precursors were equally covered in all patients, we may take into consideration synthetic liver function. The liver is the main source of GSH exported into blood. The export of GSH and its conjugates from liver cells occurs via transporters, referred to as organic anion-transporting polypeptides. These are generally believed to carry out electroneutral exchange, in which the cellular uptake of organic anions is coupled to the efflux of anions such as HCOJ, GSH, GSSH, and/or glutathione S-conjugates [6, 20]. The production in the liver and export from it are related to GSH functions, and at least two principles may be implicated. The primary GSH function here is directed toward detoxification of injurious external agents to prevent damage to the organism. The second principle is related to high-intensity oxygen-based metabolism and detoxification of certain compounds by internal organs. We did not measure GSH levels, but its synthesis can be sufficient to indirectly assess the other substances that are exclusively synthesized in the liver. When comparing the values of albumin and fibrinogen (Table 2) no significant differences were found between the groups particularly in the monitored period, even with regard to the administration of selenium. Also, severe liver failure would lead to a decrease in CRP [23], although there was no notion of a significant decrease at any time intervals Tl, T2, and T3. On the other hand, a significant fact for persistent low GR activities must then be the availability of NADPH, as the hydrogen required for this reduction of glutathione by GR comes from NADPH arising from the pentose phosphate pathway (PFP). Since NADPH is needed to drive redox reactions as a strong reducing agent, the NADP+/NADPH ratio is kept very low [30]. Here, we must consider the glucose metabolism in sepsis, where hyperglycemia is a common feature. The reduction of glutathione reductase activity may be directly caused by glucose or glucose-6-phosphate glycation [5], The speed of glyca-tion is dependent upon the amount of open-chain sugar, in which the carbonyl group is available for Schiff base formation. We assume that this toxic effect of glucose is at least partly prevented by strict glycemic control and, as such, other possible toxic effects and inhibition of PFP alone will not be mentioned. It remains, therefore, only a possible use of NADPH. Basically, we come around in a seemingly vicious circle, as the oxidative PFP produces NADPH, feedingeither glutathione reductase to maintain glutathione in its reduced state (antioxidant) or NADPH oxidase and NO synthase for producing radicals and NO, respectively (prooxidant) [32]. This prooxidative phase is especially developed in septic patients, as the inter- nal conditions indicate the preferential use of NADPH for NADPH oxidases in phagocytic and nonphagocytic cells as well as the use of NO synthase to form radicals in cooperation with myeloperoxidase (see characteristic parameters in Table 3 also e.g., CRP and fibrinogen in Table 2). It is true that the diagnosis of severe sepsis in critically ill patients is often very difficult and changes in body temperature, heart rate, white blood cells count, and respiratory rate show low diagnostic specificity. Positive blood cultures are often caught later as the situation requires. The biochemical parameters for the aforementioned purpose are set by CRP and procalcitonin [23]. For low and irregular measurements of procalcitonin, we did not use it as a reference marker in patients enrolled in the study. To complete our account, we will argue that the rise of plasma CRP occurs after 4-6 h after the evocative stimulus, doubling every 8 h before peaking between 36 and 50 h. The average values of CRP in SIRS are around 100 mg/1, in sepsis 150 mg/1, and as high as 170 mg/1 for severe sepsis. After the disappearance of the stimulus, CRP gradually decreases, with a half-life of 19 h. If the noxa persists, CRP can be raised for a long time. The values reported in Table 2 would only confirm persisting prooxidant conditions. Generally, slower growth in GR activities when compared to GPx toward the values measured in healthy subjects also refers to the activation of another very important redox system. The thioredoxin system participates in both antioxidant defense and regulation of cellular signal transmission, transcription, cell growth, and apoptosis and its function is therefore critical for cell survival. The reaction substrate is thioredoxin, which is oxidized. The regeneration is catalyzed by selenoenzyme thioredoxin reductase, which is important to consider in terms of selenium supplementation. Thioredoxin reductase is the only known enzyme that reduces oxidized thioredoxin but the reduction requires NADPH-like GR. In view of the increased demands for protein synthesis in septic patients, it is likely that there is a preferred restorative functionality of the system not only because of its importance, but also due to the fact that it is also a selenoenzyme. As yet, there are no data about any developmental changes in GR activity, so none have mentioned selenoenzyme thioredoxin reductase in septic patients after selenium administration. For our consideration of the assessment of the clinical status of development in all four subgroups, we used a comparison of the number of blood components and ratios of neutrophils/Iymphocytes in all subgroups (Table 3). Sepsis is an obvious risk factor for thrombocytopenia in critically ill patients, highlighting that thrombocytopenia is dependent on the severity of sepsis [9], Platelets themselves are capable of producing the activation of free radicals and thus participate in the elimination of bacteria [4], A decrease in thrombocytes in septic patients is typical during the first 4 days in the ICU. The subgroups did not reveal any statistically significant differences (Table 3), which would indicate the same degree of severity of the clinical condition of the sub- ^ Springer Restoration of antioxidant enzymes in the therapeutic use of selenium in septic patients 323 40 original article groups. Compared with the reference levels, there is an increase in the total number of leukocytes in the blood to > 12,000 mm3 (leukocytosis), which is typical for a bacterial infection or a decrease < 4,000 mm3 (leukopenia) in long-term viral or bacterial infections [11], which leads to the depletion of reserves and incentive mechanisms. Statistical analysis confirmed no differences between groups in the time intervals Tl and T2 (p>0.05) in the leukocyte count, which can testify the comparable status of infection development intensity in both groups during treatment with selenium but no longer at T3. Difference between A and B is considered to be very statistically significant. Also, the number of days to reduce the number of lymphocytes was dependent on the extent of surgery or aggravated during SIRS. The average time of lymphocytopenia is 2-7 days [31]. The rapid development of changes in the population of white blood cells reflects the immune system's response to postoperative stress, SIRS, and sepsis [24]. A correlation was found between the severity of clinical status and the degree of neutrophilia and lymphocytopenia. The ratio of neutrophils and lymphocytes simply and reliably portrays the severity of inflammation [31]. In our Se-AOX study, there were no statistically significant differences (p>0.05) (Table 3) between the placebo group and subgroups AI, A2, Bl, and B2 at intervals Tl, T2, and T3 when comparing neu-trophils/lymphocytes ratios. In conclusion, the comparison of routine biochemical, hematological, and coagulation parameters between the groups and subgroups did not show any statistically significant differences, which is comparable with the results of several international multícenter randomized studies. An increase in GPx activity occurred after initiation of selenium supplementation. We consider the major finding of our study that, even after treatment with selenium, the antioxidant enzyme activities in patients do not reach the level of those of healthy people. An increase in GPx was observed in all groups and subgroups, but pronounced in subgroups containing surgical patients. This raises the question of the use of higher therapeutic daily doses of selenium, which could be up to twice the dose used in this study. The benefits of using a bolus of sodium selenite pentahydrate are arguable at the beginning of therapy, as is the use of its prooxidant effect in the initial phase of sepsis, though rather for the nonsurgical group of patients. Surgical patients, on the other hand, are assumed to be able to manage an acute increase in oxidative stress thanks to the intervention, which the body usually responds to immediately. That being said, recent studies dealing with the issue of administration of high doses of selenium have not yet produced conclusive results. SOD activity has been shown to be a good and relatively quick indicator of a decrease in superoxide radical formation when monitoring the effectiveness of the selected treatment. Monitoring GR activity appears to be meaningful and informative for considering patient success. We therefore consider GR when considering elective treatment in relation to its physiological and biochemical importance and possibility of inactivation. Based on the results obtained by us in the Se-AOX study we can assume that even if supplementation of sodium selenite pentahydrate does not belong to the "live-saving therapy" it could help to balance oxidative stress during systemic inflammation reaction, emphasizing on the efficiency of the dose used in surgical patients. It is still necessary to collect and analyze the results for a better indication of drug administration in various disease states which require further clinical studies. Acknowledgements Work was supported from Slovak grant agency for Science VEGA no. 1 /1236/12 and 1 /0624/08. Ethics committee of Pavol Jozef Šafárik University in Košice approved the study 109/2011. Conflict of interest None of the authors has any financial/commercial conflicts of interest with the published data. References 1. Andrews PJ, Avenell A, Noble DW, Campbell MK, Croal BL, Simpson WG, Vale LD, Battison CG, Jenkinson DJ, Cook JA, Scottish Intensive care Glutamine or seleNium Evaluative Trial Trials Group. Randomized trial of glutamine, selenium, or both, to supplement parenteral nutrition for critically ill patients. BMJ. 2011; 342:dl542, doi:10.1136/bmj. dl542. 2. Angstwurm MW, Engelmann L, Zimmermann T, Lehmann C, Spes CH, Abel P, Strauss R, meier-Hellmann A, Insel R, Radke J, Schüttler J, Gärtner R. 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OATP8/lB3-medi-ated cotransport of bile acids and glutathione: an export pathway for organic anions from hepatocytes? J Biol Chem. 2006;281(41):30326-35. 7. Chan WS, Dedon CP. The biological and metabolic fates of endogenous DNA damage products. J Nucleic Acids. 2010;13, Article ID 929047, doi:10.4061/2010/929047. 8. de Menezes CC, Dorneles AG, Sperotto RL, Duarte MM, Schetinger MR, Loro VL. Oxidative stress in cerebrospinal fluid of patients with aseptic and bacterial meningitis. Neurochem Res. 2009;34(7):1255-60. 9. Faviere W, Boechat T. Sepsis: thrombocytopenia is bad, not recovering thrombocytopenia is too bad. Crit Care. 2011;15(Supp 1):P440. 324 Restoration of antioxidant enzymes in the therapeutic use of selenium in septic patients £l Springer 41 original article 10. Finley JW, Duffleld A, Ha P, Vanderpool RA, Thomson CD. Selenium supplementation affects the retention of stable isotopes of selenium in human subjects consuming diets low in selenium. Br J Nutr. 1999;82(5):357-60. 11. Firment J, Hudak V, Grendel T. Difficulties in implementation of the recommendations for the diagnosis and treatment of severe sepsis and sepsis shock. Anesteziologie a Intenzivní Medicína 2008;19(5):252-9. 12. Forceville X, Laviolle B, Annane D, Vitoux D, Bleichner G, Korach JM, Cantais E, Georges H, Soubirou JL, Combes A, Bellissant E. Effects of high doses of selenium, as sodium selenite, in septic shock: a placebo-controlled, randomized, double-blind, phase II study. Crit Care. 2007;11(4):R73. 13. Geoghegan M, McAuley D, Eaton S, Powell-Tuck J. Selenium in critical illness. Curr Opin Crit Care. 2006;12(2}:136-41. 14. Hardy G, Hardy I. Selenium: the Se-XY nutraceutical. Nutrition. 2004;20(6):590-3. 15. Heyland D. Selenium supplementation in critically ill patients: can too much of a good thing be a bad thing? Crit Care. 2007;11(4):153. 16. Ince C. The microcirculation is the motor of sepsis. Crit Care. 2005;9(Supp 4):13-9. 17. Kharb S, Singh V, Ghalaut PS, Sharma A, Singh GP. Role of oxygen free radicals in shock. J Assoc Physicians India. 2000;48(10):956-7. 18. Kuklinski B, Zimmermann T, Schweder R. Decreasing mortality in acute pancreatitis with sodium selenite. Clinical results of 4 years antioxidant therapy. Med Klin (Munich). 1995;90 (Supp 1):36-41. 19. Levy MM, Fink MP, Marshall JC, Abraham E, Angus D, Cook D, Cohen J, Opal SM, Vincent JL, Ramsay G. 2001 SCCM/ ESICM/ACCP/ATS/SIS International Sepsis Definitions Conference. Intensive Care Med. 2003;29:530-8. 20. Mahagita C, Grassl SM, Piyachaturawat P, and Ballatori N. Human organic anion transporter 1B1 and 1B3 function as bidirectional carriers and do not mediate GSH-bile acid cotransport. Am J Physiol Gastrointest Liver Physiol 2007;293(l):G271-8. 21. Manzanares W, Biestro A, Torre MH, Galusso F, Fac-chin G, Hardy G. High-dose selenium reduces ventilator-associated pneumonia and illness severity in critically ill patients with systemic inflammation. Intensive Care Med. 2011;37(7):1120-7. 22. Manzanares W, Hardy G. The role of prebiotics and syn-biotics in critically ill patients. Curr Opin Clin Nutr Metab Care. 2008;ll(6):782-9. 23. Meisner M. Biomarkers of sepsis: clinically useful? Curr Opin Crit Care. 2005;ll(5):473-80. 24. Menges T, Engel J, Weters I, Wagner RM, Little S, Ruwoldt R, Wollbrueck M, Hempelmann G. Changes in blood lymphocyte populations after multiple trauma: association with posttraumatic complications. Crit Care Med. 1999;27(4):733-40. 25. Mishra V, Baines M, Perry SE, McLaughlin PJ, Carson J, Wenstone R, Shenkin A. Effect of selenium supplementation on biochemical markers and outcome in critically ill patients. Clin Nutr. 2007;26(l):41-50. 26. Rayman MP. The importance of selenium to human health. Lancet, 2000;356(9225):233-41. 27. Ritter C, Andrades M, Frota Junior ML, Bonatto F, Pinho RA, Polydoro M, Klamt F, Pinheiro CT, Menna-Barreto SS, Moreira JC, Dal-Pizzol F. Oxidative parameters and mortality in sepsis induced by cecal ligation and perforation. Intensive Care Med. 2003;29(10):1782-9 28. Salama A, Sakr Y, Reinhart K. The role of selenium in critical illness: Basic science and clinical implications. Indian J Crit Care Med. 2007;ll(3):127-38. 29. Valenta J, Brodska H, DrabekT, Hendl J, KazdaA. High-dose selenium substitution in sepsis: a prospective randomized clinical trial. Intensive Care Med. 2011;37(5):808-15. 30. Wollin MS, Ahmad M, Gao Q, Gupte SA. Cytosolic NAD(P) H regulation of redox signaling and vascular oxygen sensing. Antioxid Redox Signal. 2007;9(6):671-8. 31. Zahorec R. Ratio of neutrophil to lymphocyte counts-rapid and simple parameter of systemic inflammation and stress in critically ill. Bratisl Lek Listy. 2001;102(1):5-14. 32. Zuurbier CJ, Eerbeek O, Goedhart PT, Struys EA, Verho-even NM, Jakobs C, Ince C. Inhibition of the pentose phosphate pathway decreases ischemia-reperfusion-induced creatine kinase release in the heart. Cardiovascular Res. 2004;62:145-53. Springer Restoration of antioxidant enzymes in the therapeutic use of selenium in septic patients 325 42 Clinical Biochemistry 47 (2014) 44-50 ELSEVIER Contents lists available at ScienceDirect Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem Selenium adjuvant therapy in septic patients selected according to I Carrico index Ladislav Kočana, Janka Vašková b'*, Ladislav Vaško b, Jana Šimonovaa, Robert Šimonc, Jozef Firmenta a Isi Clinic of ňnaesthesiology and Intensive Care Medicine, Louis Pasteur University Hospital in Košice, Slovak Republic Department of Medical and Clinical Biochemistry, Faculty of Medicine, Pavol Jozef Šafárik University in Košice, Slovak Republic c 2nd Clinic of Surgeiy. Louis Pasteur University Hospital in Košice. Slovak Republic ARTICLE INFO ABSTRACT Article history: Received 24 March 2014 Received in revised form 3 July 2014 Accepted 4 July 2014 Available online 12July 2014 Keywords: Oxidative stress Oxygenation index Renal functions Selenium Sepsis Objectives: The objective of this paper is to highlight the selected group of patients in which adjuvant therapy seems to have a more pronounced positive effect. Design and methods: 65 septic patients from the prospective observational study Se-AOX (2008-2012) (ClinicalTrials.gov Identifier: NCT02026856) were divided into a Se group, receiving sodium selenite in a continual infusion of 750 ug/24 h for 6 days, and a placebo group. They were subsequently divided into subgroups according to the initial Carrico index (CI) on the day of admission: CI > 200 and CI < 200. Dynamical changes in glutathione peroxidase (CPx), glutathione reductase (GR) and superoxide dismutase activities were recorded at two day intervals. Clinical parameters and mortality were compared. Results: The CI increased in subgroup Se-CI < 200 with negative correlation against subgroup Placebo-CI < 200 during the last measuring period (p < 0.02). GPx activity increased in selenium subgroups with negative correlation against placebo subgroups (p < 0.01). SOD activity was elevated in all subgroups in comparison with values of healthy subjects. Conclusions: Adjuvant selenium therapy seems to be beneficial for a selected group of patients with acute lung injury. However, as is clear from the results discussed, this is not the case with persistent renal failure, as this leads to an inability to maintain synthetic renal function and ensure GPx synthesis. © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Introduction Sepsis is associated with systemic mediator-induced alterations to oxygen utilization, including increased oxygen demand, altered oxygen extraction, and decreased myocardial contractility [ 1 ]. When developing global respiratory insufficiency and hypoxia, where the Carrico index—ratio of partial pressure arterial oxygen and fraction of inspired oxygen (Pa02/Fi02) is less than 280, it is necessary to intubate the patient and start artificial ventilatory support. Acute lung injury (ALI) is a multifactorial problem. The basis for establishment may be interstitial pulmonary edema, dystelectases, fluidothorax or increased pulmonary shunts. At the inception of acute respiratory distress syndrome (ARDS) with a CI less than 200, special protection is required in the form of mechanical ventilation [2]. A significant increase in the fraction of inspired oxygen is required in cases of worse tissue oxygenation. These factors are the cause of worsening CI and further damage to the lung tissue [3]. * Corresponding author at: Department of Medical and Clinical Biochemistry. Faculty of Medicine, Pavol Jozef Šafárik University in Košice, Tr. SNP 1, 040 66 Košice, Slovak Republic. E-mail address: janka.vaskova@upjs.sk (J. Vašková). Hawker et al. [4] presented that selenium (Se) deficiency can augment pulmonary injury from high concentrations of inspired oxygen. Septic patients have a significantly reduced level of plasma Se with antioxidant, anti-inflammatory and immunological functions 15-7], and are exposed to mechanical ventilation with high concentrations of inspired oxygen at the same time. The discovery of glutathione peroxidase (GPx) in the cytosol lent support to the protective role of Se. Se is an essential component of GPx which acts synergistically with tocopherol in the regulation of lipid peroxidation. In tandem with catalase, it degrades hydrogen peroxide to water (or corresponding alcohols) via glutathione reductase and flavin adenine dehydrogenase (FAD) in the pentose phosphate shunt [8]. Several clinical studies have been devoted to the issue of selenium antioxidant therapy in selected pathologies [5,9-11 ]. Individual studies addressing this issue are varied in respect to the dose and duration of selenium administration, and the form of administration (bolus vs. continuous administration) and show conflicting results. However, other studies were also published later which demonstrated no statistically significant reduction in mortality in any groups [ 12,13]. Despite findings from previous studies that plasma GPx is an easily renewable selenoprotein, and that other components of blood containing GPx, like erythrocytes and platelets, are still required to restore long-term http://dx.doi.org/] 0.1016/j.dinbiochem.2014.07.004 0009-9120/© 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. L Kočan et aí. / Clinical Biochemistry 47 (2014) 44-50 45 supplementation [ 14], selenium supplementation is still not advised "as standard" to the critically ill. Considering the above-mentioned pathophysiological importance of Se metabolism, and especially its antioxidant role in lung injury, we (within Se-AOX study from 2008 to 2012) purposefully selected a group of patients to test the hypothesis that the selected dose of selenium would decrease the severity of the disease, and improve Carrico index. Materials and methods From January 2008 to December 2012 at Clinic I of Anaesthesiology and Intensive Care Medicine, University Hospital of Louis Pasteur in Košice, Slovak Republic a prospective observational study, Se-AOX, was conducted to look at selenium supplementation. This study looked at parenteral administration of selenium to septic patients and subsequently monitored dynamic changes of selected activities of antioxidant enzymes and the development of clinical status. The university ethics committee approved the study 109/2011, ClinicalTrials.gov Identifier: NCT02026856. 72 patients were enrolled in the whole Se-AOX with an APACHE II score between 19 and 24 points at ages between 23 and 79 years old who developed sepsis, severe sepsis or septic shock during hospitalization as defined by the International Sepsis Definitions Conference [ 15 ]. A SOFA score was calculated at the time periods 1 -2 days (Tl),3-4 days (T2) and 5-6 days (T3) according to an internet calculator (http://www.sfar.org/scores2/sofa2.php), and more detailed information about criteria for the diagnosis of sepsis is dealt with by Vašková et al. [16]. 65 patients were included in the evaluation (Table 1). The study was conducted only in the Louis Pasteur University Hospital. Due to the severity of the condition, the selection criteria could not include more patients for that period. Regarding the ventilation period, we excluded patients who had received mechanical ventilation for less than 24 h. We had supposed shorter exposure of oxygen radicals in the air mix with artificial ventilation. Neuromuscular disease and terminal illness were also excluding criteria; see flow-diagram (Fig. 1). The CI as Pa02/ Fi02 (partial pressure of oxygen in arterial blood/fraction of inspired oxygen) ratio in patients was determined daily. Patients were divided into two groups. Se-group consisted of 31 patients, who received selenium supplementation during hospitalization in the form of sodium sefenite pentahydrate at 750 mg/day for 6 days as a continual infusion immediately after admission to our department (1000 mg of sodium selenite pentahydrate = 333 ug of selenium) (Selenase, Vivax; selenium hereinafter). At the same time, alanyl glutamine solution was administered via a central venous catheter at a daily dose 100 ml (2g) for 6 days. The placebo group consisted of 34 patients who received continuous saline NaCI 50 ml/day for 6 days as a continuous infusion (excluding additional infusion therapy). Patients were further divided into subgroups according to CI on the day of admission to ICU. 200 was chosen as the threshold CI value. The nutritional strategy (enteral vs. parenteral, qualitative and quantitative characteristics of the diet) was dependent on the patient's diagnosis and nutritional status, as well as the standard therapeutic approaches to the disease. Some of the patients were without any enteral or parenteral nutrition longer than 48 h (8 patients in selenium and 10 patients in non-selenium group). The reason of this strategy was the presence of the acute phase of septic shock. Counts of this patients in both groups were similar and without statistical significant differences in mortality when compared patients with enteral and parenteral nutrition. Food administration was cyclical, to stimulate the diurnal rhythm. The daily dosage of glucose ranged from 1.5 g/kg up to 5.5 g/kg. The total daily supply of amino acids ranged from 0.8 to 2.5 g/kg (0.5 to 0.75 g/kg for renal and liver injury, from 0.7 to 1.0 g/kg in stable patients, 1.0 to 1.5 g/kg in a poor state of nutrition and catabolism, and 1.5 to 2.0 g/kg for severe malnutrition). Enteral nutrient intake (Nutrison-Multifibre) was provided via naso-gastric tube and parenteral nutrition solutions (SmofKabiven and Aminomix II) were administered intravenously. Fat substitution (Smoflipid and Omegaven) was performed after the acute stage of the disease subsided and was administered at 0.5 g/kg/day. All groups were also administered antioxidants parenter-ally: ascorbic acid 1000 mg/24 h, tocopherol 30 mg/24 h, 100 mg/24 h thiamine, 100 mg/24 h pyridoxine and 1000 f.ig/24 h cobalamin. The determination of biochemical and hematological parameters of blood samples formed part of the routine diagnostic methods. Data collection for the Se-AOX study was performed at two-day intervals: Tl (lst-2nd day), T2 (3rd-4th day) and T3 (5th-6th day). Kinetic methods for estimating the activities of glutathione peroxidase (GPx. E.C. 1.11.1.9) and glutathione reductase (GR, E.C.1.6.4.2) were performed using a kit (Sigma-Aldrich, Germany) and that of superoxide dismutase (SOD, E.C. 1.15.1.1) by means of the SOD-Assay Kit-WST (Fluka, Japan) following the user manual provided. Descriptive statistics methods were used to evaluate the results. Differences between continuous variables were analyzed by a non-parametric Mann-Whitney test. Statistical significance was determined by Pearson's Cfn-square of selenium vs. non-selenium groups from the baseline to the 6-7th day. Possible benefits from the treatment were calculated by NNT test. Results There were no statistically significant differences at the time intervals Tl, T2 and T3 (p > 0.05) when comparing the plasma levels of total protein, albumin, CRP and fibrinogen, lactate, creatinine and urea between subgroups: Se-CI > 200 vs Placebo-CI > 200 and Table 1 Demographic characteristics of the patients in groups with selenium adjuvant therapy divided according to Carrico index (Ci) under or above 200 and placebo groups with oxygenation index under or above 200. Characteristics Se-CI > 200 (min-max) med Placebo-Cl > 200 (min-max) med Se-CI < 200 (min-max) med Placebo-CI <200 (min-max) med N 14 I«) 17 15 Age (23-78)53 (30-77)60 (28-68)47 (40-79)53 Gender (M/F) 7/7 12/7 12/5 12/4 APACHE score (19-40)23 (19-38)23 (19-39)25 (20-36)28 SOFA score in Tl (6-14) 11 (6-14)9 (5-14)10 (6-13)9 Severe sepsis 10 12 7 9 Septic shock 4 7 8 6 Disseminated intravascular coagulation 2 2 1 2 Bronchopneumonia 9 7 12 9 Surgical performance 9 11 12 9 Nosocomial infections'1 11 14 12 10 Dialysis 8 5 9 6 -CWH/CRRTb 5 and more days 5 2 3 1 J Cultivated pathogens: Staphylococcus aureus, Enterococcus sp., Candida sp.. Pseudomonas aeruginosa (bloodstream), Pseudomonas aeruginosa. Staphylococcus aureus. Acinetobacter sp.. Klebsiella pneumonia (respiratory tract), £ col), Candida sp., Pseudomonas aeruginosa (urinary tract). b CWH/CRRT—Continuous Veno-Venous Hemofiltration/Continuous Renal Replacement Therapy. 44 L Kočan et al I Clinical Biochemistry 47 (2014) 44-50 Critically ill patients with signs of sepsis, severe sepsis, septic shock .APACHE II 19-60 (n=95) Loss to follow up (n= 15) Patients with APACHE <19, >40 excluded (n-80) Patients met inclusion criteria APACHE 19-40 Lost to follow-up (n-S) (died in day after admission) Loss to follow7 up (n~ 7) (receiving mechanical ventilation 200 (n- 19) Placebo-CI<200 (n-15) Difference in CT development Fig. 1. Flow diagram. Patients were reselected according to Carrico index and selenium administration. Worse Carrico index indicates reduced pulmonary function and dysfunction of alveolar-capillary membrane. The Carrico index was measured every day during the whole period of study observation. Patients receiving mechanical ventilation for <24 h were excluded. Other excluding criteria were neuromuscular disease and terminal illness. sub-Se-CI < 200 vs Placebo-Cl < 200 (Table 2). The plasma levels of measured parameters showed statistical similarity. Median values of creatinine were also comparable, but with very high maximum values ensured in groups with CI > 200. Compliance to these conditions is important for the antioxidant response followed, including sufficient amounts of amino acids supplemented for the formation of glutamine and protein components of antioxidant enzymes. Comparing the number of white blood cells, neutrophils, lymphocytes, and platelets (Table 2), a statistically significant decrease was found in total white blood cell count at time T3 between subgroups Se-CI > 200 vs Placebo-Cl > 200 favoring the placebo subgroups (p < 0.05). There were no statistically significant differences at the time intervals T1, T2 and T3 when comparing other blood elements (p > 0.05). This fact testifies to the similarity of groups in the study intervals and the same degree of severity of the clinical status between subgroups. Comparing the CI (Fig. 2) in individual subgroups, a statistically significant increase in CI was found at time T3 between subgroups Se-CI < 200 and Placebo-CI < 200 in favor of selenium subgroups (p < 0.02). A statistically significant increase in GPx activity at time T3 was found in all subgroups with selenium supplementation (p < 0.01) (Table 3). No statistically significant differences were found between subgroups at the time intervals Tl, T2 and T3 (p > 0.05) in the activities of antioxidant enzymes GR and SOD. Discussion In a prospective observational study with AOX we followed the dynamic development of biochemical and hematological parameters of sepsis and dynamic changes of antioxidant enzymes in critically ill patients (n = 65) with APACHE II (median 24, min. 19, max. 40) and SOFA (median value 9, min. 4 max. 14). There was no evidence of differences between subgroups when comparing the changes in organ dysfunction by SOFA score intervals Tl, T2, and T3 See Fig. 3. Similarly, no differences were found in mortality between Se and placebo subgroups (Table 4). The liver is the central body of metabolic excretion, metabolic, detoxifying, hematological, and hemostatic functions, and a number of L Koian et aí. / Clinical Biochemistry 47 (2014) 44-50 47 Table 2 Comparison of the levels of selected parameters in plasma over theTl-T3 day interval. Parameters (reference values) Time interval (min-max) med P (min-max) med P Se Cl 200 ľlaccbo-CI > 200 Se-CI 200 Placebo-Cl < 200 Total plasma protein Tl (38.5-61.7) 50.95 (36.0-57.5) 48.38 0.2 (36.0-77.9) 50.95 (38.5-61.7)46.8 0.60 (64.0-83.0 gl-1) T2 (34.0-62.5) 50.6 (42.2-70.0) 51.2 0.85 (34.0-70.0) 50.0 (34-68.051.0 0.76 T3 (36.3-64.7) 54.4 (28.1-77.9)49.8 0.43 (34.0-65.0) 52.4 (28-78) 49.8 0.82 Albumin 1 1 (13.0-39.0)25.95 (11.2-50.0)22.5 0.72 (15.5-38.0)23.2 (15.0-46.0)25.6 0.07 (35.0-52.0 gl-1) T2 (15.1-31.9)22.03 (14.9-39.4)21.05 0.70 (14.9-39.4)22.9 (14.3-37.2)20.7 023 T3 (14.2-39.2) 24.95 (14.9-39.4)21.05 0.80 (12.9-35.5)22.3 (12.5-39.2)24.7 0.70 CRP T1 (38.2-482.0) 180.4 (23.6-367.2) 148.8 0.43 (6.6-370.4) 192.1 (4.5-445.3) 103.4 0.33 (0.1-5.0 mg I"') T2 (12.4-428.5) 181.2 (8.4-220.4) 94.1 0.19 (38.1-280.0) 162.0 (3.1-444.5) 122.5 0.88 T3 (2.7-323.3) 129.2 (21.7-180.0)92.4 0.24 (16.0-482) 126.5 (10-318.0) 170.0 0.85 Fibrinogen Tl (0.8-8.7) 4.1 (2.0-8.4) 4.9 0.38 (1.3-9.5) 5.4 (1.9-25.0)4.1 0.31 (1.8-3.5 gr') T2 (1.2-8.9)4.7 (3.0-7.2)4.2 0.72 (2.3-7.8)5.95 (1.1-14.0) 6.1 0.77 T3 (1.0-9.0)4.75 (2.0-8.4) 4.9 0.60 (2.5-10.8) 5.2 (1.3-12.0) 5.1 0.97 Lactate "IT (1.3-6.2)3.0 (1.0-6.3) 3.2 0.98 (1.4-6.2)2.0 (1.1-12.9) 2.9 0.08 (0.6-2.1 mmol r') T2 (0.9-4.3) 1.85 (1.1-7.2)2.1 0.87 (1.1-5.9)2.2 (0.9-7.2) 2.2 0.85 T3 (1.0-3.8) 1.7 (1.1-5.8) 1.6 0.24 (1.0-6.9)2.1 (1.2-4.7) 2.3 0.30 Creatinine Tl (41.2-689.6) 145 (62.1-601.1) 149 0.9 (71.9-370.5) 214.1 (42.8-315.3) 110 0.33 (53-115 unroll-') T2 (44.9-375.8) 126.5 (53.4-298.7) 109.4 0.73 (57.0-318.7) 142.4 (56.1-446) 185.3 0.91 T3 (36-649) 123.5 (57-213) 104.1 0.65 (87-312) 130 (75.4-391.5) 128.6 0.29 Blood urea nitrogen T1 (2-44)12 (2-46)11 0.36 (5-29)13 (3-37)6 0.15 (2.6-8.1 mmol I"') T2 (6-35)10 (3-32) 13.4 0.45 (2-41)15 (3-32)11 0.85 T3 (3-44)12.5 (5-25) 14.5 0.78 (1-30)11 (5-28)11 0.89 WBC Tl (5.6-32.2) 13.1 (5.1-62.2) 11.1 0.92 (3.8-25.2) 17 (0.9-25)11.9 0.18 (4.0-10.0 x lo" r1) T2 (3-31.3) 11.45 (8-34.9) 13 0.46 (5.5-21.6) 12.4 (0.1-29.3) 9.4 0.20 T3 (1.5-27.4) 13.1 (1.7-18.6) 8.9 0.05 (6.3-23) 13.4 (3.5-21.2) 11.6 0.24 Neutrophils I l (3.0-32.5) 12.75 (4.1-28.1) 10.1 0.57 (3.2-21.4) 15.3 (0.8-27.2) 9.4 0.33 (33-80%) T2 (2.6-29.6) 12.8 (5.8-24.3) 9.7 0.60 (3.7-18.3) 10 (0.9-24.9) 9.6 0.74 T3 (1.3-21.9) 10.9 (3.3-16.3) 8.9 0.36 (3.7-17.8) 12 (1.1-20.1) 9.2 0.43 Lymphocytes Tl (0.5-8.2) 1.1 (0.1-2.6) 1.0 0.76 (0.3-2.3) 1.0 (0.0-2.3) 0.9 0.43 (15-35%) T2 (0.3-8.2) 0.9 (0.4-1.9)1.2 0.26 (0.2-2.2) 1.4 (0.1-2.3)0.9 0.33 T3 (0.1-9.1)0.7 (0.6-2.0) 1.1 0.31 (0.2-3.6) 0.9 (0.1-1.8) 1.1 0.89 Platelets Tl (22-499) 178 (31-390)128 0.82 (47-410) 199 (6.6-550) 163 0.38 (150-350 x 10a]-') T2 (31-430)137 (35-429)120 0.79 (28-432)221 (12.5-445) 113 0.20 T3 (48-337) 145 (17-477)123 0.36 (13-483)231 (49-426.5) 136 0.45 other important functions. In the early stage of hepatic failure there is higher degradation of proteins by predominantly catabolic pathways. There is an acceleration in the inflammatory response, an increase in inflammatory mediators and increase in the production of acute phase proteins being released into the circulation. Through examination of non-specific inflammatory parameters fibrinogen and C-reactive protein at time intervals Tl, T2 and T3, we found a statistical similarity between groups (p > 0.05) with generally elevated levels. Under favorable conditions, the physiological function of liver tissue is restored and the damaged liver lobules are repaired ad integrum. The excessive action of noxa may exceed the compensation limits and become irreversible, resulting in hepatic failure |5]. This leads to a decrease in protein synthesis, which results in decreased levels of total protein and manifests mainly as a decrease in plasma albumin, prealbumin, transferrin, transcortin and retinol-binding protein [ 17]. The same statistical similarity was recorded between groups when comparing low plasma levels of albumin, and total protein (p > 0.05). Although, hypoalbuminemia is quite common in critically ill patients, especially in severe sepsis, on the one hand it is interpreted as a normal compensatory mechanism when inflammatory mediators increase vascular permeability to promote escape of circulatory albumin into extravascular space which may provide protection from oxidative stress [ 18 ]. On the other hand, however, there is also the possibility of reduced production by the liver, which may be due to the persistent lack of capacity in the plasma compartment that influences the development of illness and outcome of patients. Hepatic failure confirms, in addition to clinical signs of kidney damage, serum creatinine and blood urea nitrogen levels (Table 2). Creatinine, however, is also formed by cleavage of phosphocreatine in the muscles, but the increase in serum creatinine can be further increased by reduced renal excretion. But both are removed by dialysis, and a number of patients from each group underwent dialysis (at least 5 patients in the placebo-Cl > 200 and not more than 9 in Croup Se-CI < 200) (Table 1). Monitoring non-specific inflammatory parameters and proteo-synthetic processes is a very complex process, and especially difficult to evaluate in the specific pathophysiology of organ dysfunction. The interpretation of results is also hampered by the diversity of applications and types of artificial nutrition. Although. Montejo et al. [19] pointed out that diet enriched with pharmaconutrients in patients requiring enteral nutrition could have beneficial effects. A diet enriched with omega-3/omega-6 fatty acids and elevated antioxidants led to improvements in clinical outcomes of patients with ALI/ARDS [20]. An assessment of the function these organs have on the metabolism of therapeutic agents like selenium is of particular importance. Septic patients have a significantly reduced level of plasma selenium with antioxidant, anti-inflammatory and immunological functions [6,7]. This is directly related to their high mortality rates. It was observed that Se (in the form of sodium selenite or selenium methionine) travels in one of three directions, into plasma without being taken up by liver; via the hepatopancreatic subsystem, or alternatively, via the lymphatic system into the plasma; or taken up by the liver. From there, some is returned to the intestine via pancreatic secretion of bile, some enters plasma, and the rest enters a pool considered as tissues. From this pool, selenium moves through delay compartments into the plasma or into other tissue pools, including red blood cells. From the plasma, Se returns to the liver, tissues, or urine |21 ]. Following absorption, the liver appeared to extract 50% of the Se, with the remainder staying in the plasma or entering the lymph [21]. According to the determined parameters, liver function is impaired and Se cannot be expected to be incorporated into forms of transport (selenoproteins P) or bound to albumin bound with sufficient tissue redistribution. GPx is also a selenoenzyme, which is also an important part of the antioxidant mechanism of glutathione. From the data measured in Tl (Table 3) it is evident that its activity in all groups is very low when compared to the values measured in healthy volunteers [16]. L Kočan et al I Clinical Biochemistry 47 (2014) 44-50 Placebo-CI>200 Placebo-CI<200 ii (f 7 b SOFA Placebo-CI>200 Iii *i i Fig. 2. Comparison of CI groups with a placebo. A statistically significant increase in CI was found when comparing oxygenation indexes at timeT3 between subgroups Se-CI < 200 vs F'S- 3- The dynamic development of SOFA- score in patients divided into subgroups ac-Placebo-Cl < 200 in favor of selenium subgroups ("p < 0.02). cording to C! on the day of admission to 1CU. After six days of Se supplementation atT3, there were significant differences in GPx activities in both Se groups in favor of Se groups when compared to the placebo. This would clearly refute impaired liver function, as the activity of GPx isoforms are regulated by the Se status. This essentially eliminated the possibility of comparable conditions for biochemical parameters in all other groups. Kidney proximal tubular cells are the main source of GPx activity in the plasma [22], El-far etal. [23) found a highly significant negative correlation between GPx activity in plasma and serum creatinine, and also blood urea nitrogen in patients with renal impairment or chronic renal failure on hemodialytic maintenance. Each group included a number (usually less than half) of patients who were on dialysis (Table 1) and, of those, some required Continuous Veno-Venous Hemofiltration/Continuous Renal Replacement Therapy (CWH/CRRT) for 5 days or more, indicating renal impairment or chronic renal failure throughout the period of selenium administration. Due to the reasons mentioned above, the last subgroup can be considered confusing, as the Se adjuvant therapy is ineffective in this case. GPx uses glutathione as the reducing agent to convert substrate peroxides to water and oxidized glutathione is recycled back to glutathione by Table 3 Comparison of plasma levels of antioxidant enzymes in selenium and placebo groups. Parameters Time (min-max) med P (min-max) med P interval Se-CI > 200 Placebo-CI » 200 Se-CI < 200 Placebo-CI < 200 GPx Tl (0.02-3.28) 0.19 (0.11-1.89) 0.21 0.96 (0.02-1.66)0.16 (0.08-1.32) 0.12 0.71 Oikatr1) II (0.01-2.98) 0.30 (0.01-1.22) 0,21 0.71 (0.12-2.67)0.40 (0.12-1.77) 0.21 0.66 rs (0.10-1.23) 0.52 (0.01-0.59) 0.14 0.01 (0.18-2.97)0.53 (0.05-2.02) 0.09 0.0Í CR Tl (0.03-1.77) 0.33 (1.01-3.11) 1.31 0.76 (0.04-1.78)0.61 (0.18-1.99)0.48 0.87 (ujtatr') 12 (0.02-4.95) 0.61 (0.08-1.42) 0.42 0.61 (0.03-2.95) 0.87 (0.05-2.22) 0.57 0.18 T3 (0.02-2.82) 0.50 (0.08-2.27) 0.59 0.81 (0.02-4.60) 1.18 (0.03-1.27) 0.61 0.37 SOD Tl (3.43-6.45)4.51 (3.21-6.15)4.48 0.83 3.50-8.89) 4.41 .42-9.01)4.38 0.39 (jikatr') T2 (3.34-6.05)4.01 (2.98-5.74) 3.98 0.67 (3.26-6.12)4.34 (3.36-6.01)3.84 0.09 T3 (3.15-6.08)4.5 (3.11-5.34)4.21 0.43 (3.25-6.19)4.17 (3.05-5.23) 3.76 0.40 L Kočan et al. / Clinical Biochemistry 47 (2014) 44-50 49 Table 4 Mortality of patients divided into groups according to CI > 200 and CI < 200 on the day of admission (Tl). Group of patients Mortality Total Statistics Survived Died Se-Ci > 200 7 7 14 X2 = 0.022 Placebo-CI > 200 9 10 19 p = 0.88 Total 16 17 33 NNT = 39 Se-CI < 200 10 7 17 X2 = 2.079 Placebo-CI < 200 5 10 15 p = 0.15 Total 15 17 32 NNT = 4 GR. There were no statistically significant differences between groups; however, there is an obvious upward trend in the activity of GR in Se groups. In Se-Ci < 200, the values are comparable with a group of healthy volunteers [16]. SOD activity was identified a survival predictor for patients for about 3 h after the development of sepsis [24]. Higher SOD activity was observed in patients with septic shock. Kharb et al. [25] and Bela et al. [26] showed that patients with a low level of SOD had a higher mortality. In our study groups, however, the mean input SOD activity values were found to be elevated when compared to the activity in healthy volunteers in all subgroups [ 16]. An activation of extracellular SOD has a strict dependence on reduced glutathione or a low redox potential of the cell [27]. This phenomenon may be related to the higher production of superoxide radicals resulting from the greater oxidative stress in septic patients, which requires an increase in elimination, but also leads to higher peroxide formation, whose elimination is dependent on the activity of peroxidases. As antioxidant enzymes commonly use the oxido-reduction properties of metal cofactors for catalytic activity, there is also the possibility for supplementation redox active trace elements in critical illness [28]. In the course of therapy in the Se-AOX study there was only a slight decrease in SOD activity in all four subgroups. Comparison of the number of leukocytes revealed a statistically significant decrease in total white blood cell count at time T3 between subgroups Se-CI > 200 vs Placebo-CI > 200 in favor of the placebo (p < 0.05). Severe sepsis is characterized by a rise in the total number of leukocytes in the blood of > 12 000 mm3. This is typical of bacterial infection, while a decrease <4000 mm3 is typical of viral infections or prolonged bacterial infections [29], leading to the depletion of reserves and incentive mechanisms of the body. In our study, Se-AOX we found a remarkable and statistically significant improvement in respiratory function, quantified on the basis of CI index. A statistically significant increase in CI was found between the subgroups when comparing the CI at time 13 between subgroups Se-CI < 200 and Placebo-CI < 200 in favor of subgroups supplemented with selenium (p < 0.05). Hawker et al. [4] showed that manifestation of pulmonary oxygen toxicity was more severe in Se deficient animals. Furthermore, the findings of higher lung Se concentration after exposure to 95% 02 for 36 h than after air exposure in Se supplemented animals suggest that Se is redistributed into the lungs in response to oxidative stress. This will take a more positive meaning for significantly higher values of GPx, which are found in groups supplemented with Se. Conclusions It can be assumed that the lung tissue in bronchopneumonia ARDS is exposed to higher oxidative stress, and thus the effect of reactive oxygen species generated endogenously and exogenously is higher. This is especially true when administered to high fractions of inspired oxygen Fi02 during mechanical ventilation. This seems to account for the most serious damage to the lung parenchyma, as the most intense stress reaction takes place there. It also offers the greatest potential target for therapeutic antioxidant molecules. The subgroup with lower CI had improved pulmonary functions after selenium administration. Selenium administration appeared to be beneficial in these cases. We expect that the percentage would have been even higher if it had not been for the proportion of patients that had undergone continual renal replacement therapy for more than 5 days. This therapy prevented the synthetic function of GPx creation from selenium administration. Conflict of interest None of the authors has any financial/commercial conflicts of interest with the published data. Acknowledgments The study was supported by grants of Scientific Grant Agency of the Ministry of Education, Science, Research and Sport of the Slovak Republic nos. 1/0799/09 and 1/1236/12. References 11J Vincent JL The international sepsis forum's frontiers in sepsis: high cardiac output should be maintained in severe sepsis. Crit Care 2003;7:276-8. [2] Dellinger RP. Levy MM. Carlet JM, Bion J. Parker MM. Jaeschker R, et al. Surviving sepsis Campaign: international guidelines for management of severe sepsis and septic shock: 2008. 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Laviolle B, Annane D, Vitoux D, Bleichner G. Korach JM, et al. Effects of high doses of selenium, as sodium selenite. in septic shock: a placebo-controlled, randomized, double-blind, phase II study. Crit Care 2007;11;R73. [10] Heyland D. Selenium supplementation in critically ill patients: can too much of a good thing be a bad thing? Crit Care 2007; 11:153. [11] Kuklinski B, Zimmermann T, Schweder R. Decreasing mortality in acute pancreatitis with sodium selenite. Clinical results of 4 years antioxidant therapy. Med Klin (Munich) 1995;90:36-41. [12| Andrews PJ, Avenell A. Noble DW, Campbell MK, Croal BL, Sipmson WG, etal. Scottish Intensive care Clutamine or seleNium Evaluative Trial Trials Group. Randomised trial of glutamine, selenium, or both, to supplement parenteral nutrition for critically ill patients. BMJ 2011;342:dl542. http://dx.doi.Org/l0.1136/bmj.dl542. [13] Valenta j, Brodská H, Drábek T, HendlJ, Kazda A. 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J Chin Med Assoc 2009;5:225-6. [19] MontejoJC, Zarazaga A. Lopez-Martinez J, Urrútia G, Roqué M, Blesa AL, et al. Immunonutrition in the intensive care unit. A systematic review and concensus statement. Clin Nutr 2003;22:221-3. [20] Pontes-Arruda A, Demichele S, Seth A. Singer P. The use of an inflammation-modulating diet in patients with acute lung injury or acute respiratory distress syndrome: a meta-analysis of outcome data.J Parenter Enter Nutr 2008;32:596-605. [21 ] Wastney ME, Combs Jr GF. Canfield WK. Taylor PR. Patterson KY. Hill AD, et al. A human model of selenium that integrates metabolism from selenite and selenomethionine. J Nutr 2011:141:708-17. [22] Avissar N, Omt D, Yagil Y. Horowitz S. Watkins RH. Kerl EA. et al. Human kidney proximal tubules are the main source of plasma glutathione peroxidase. Am J Physiol 1994;266:C367-75. 50 L Kocan et al I Clinical Biochemistry 47 (2014) 44-50 (23] El-far MA, Bakr MA, Farahat SE. Abd El-Fattah EA. Glutathione peroxidase activity in patients with renal disorders. Clin Exp Nephrol 2005;9:127-31. |24] Ritter C, Andrades M, Frota Junior ML, Bonatto F, Pin ho RA, Polydoro M, et al. Oxidative parameters and mortality in sepsis induced by cecal ligation and perforation. Intensive Care Med 2003;29:1782-9. ]25] Kharb S, Singh V. Ghalaut PS, Sharnia A, Singh GP. Role of oxygen free radicals in shock. J Assoc Physicians India 2000;48:956-7. |26| Bela P, Ball! R, Sane AS, et al. Oxidative stress status: possible guideline for clinical management of critically ill patients. Panminerva Med 2001;43:27-31. [27] Carroll MC, Giroaurd JB, Ulloa JL. Subramaniam JR, Wong PC, Valentine JS, et al. Mechanisms for activating Cu- and Zn-containing superoxide dismutase in the absence of CCS Cu chaperone. Proc Natl Acad Sci U S A 2004; 101:5964-9. [281 Rech M.ToUTovbin A,SmootT, MlynarekM. Heavy metal in the intensive care unit: a review of current literature on trace element supplementation in critically ill patients. Nurr Clin Pract 2014;29:78-89. [291 Zahorec R, FirmentJ, StrakovaJ, MiknlaJ, Malik P, Novak 1, et al. Epidemiology of severe sepsis in intensive care units in the Slovak Republic. Infection 2005;33:122-8. Publikácia: Selenium adjuvant therapy in septic patients selected according to Carrico index, bola ocenená Lekárskou Fakultou Univerzity Pavla Jozefa Šafárika v Košiciach pre Oblasť výskumu 18. Lekárske, farmaceutické a nelekárske zdravotnícke vedy ako: „Najvýznamnejšie vedecké práce zamestnancov Univerzity Pavla Jozefa Šafárika v Košiciach publikované v roku 2014 podľa fakúlt a akreditovaných oblastí výskumu" (Príloha č.l). 2.5.4 Citácie štúdie SE-AOX v Metaanalýzach a Systémových prehľadoch Výsledky štúdie Se-AOX boli doteraz citované v nasledujúcich systematických prehľadoch a metaanalýzach: 1. Manzanares W, Lemieux M, Elke G, Langlois PL, Bloos F, Heyland DK. High-dose intravenous selenium does not improve clinical outcomes in the critically ill: a systematic review and meta-analysis. CritCare. 2016 Oct 28;20(1):356. doi: 10.1186/sl3054-016-1529-5. PMID: 27788688; PMCID: PMC5084353. 2. Zhao Y, Yang M, Mao Z, Yuan R, Wang L, Hu X, Zhou F, Kang H. The clinical outcomes of selenium supplementation on critically ill patients: A meta-analysis of randomized controlled trials. Medicine (Baltimore). 2019 May;98(20):el5473. doi: 10.1097/MD.0000000000015473. PMID: 31096444; PMCID: PMC6531101. 3. Mousavi MA, Saghaleini SH, Mahmoodpoor A, Ghojazadeh M, Mousavi SN. Daily parenteral selenium therapy in critically ill patients: An updated systematic review and metaanalysis of randomized controlled trials. Clin Nutr ESPEN. 2021 Feb;41:49-58. doi: 10.1016/j.clnesp.2020.11.026. Epub 2021 Jan 19. PMID: 33487307. 4. Jaff S, Zeraattalab-Motlagh S, Amiri Khosroshahi R, Gubari M, Mohammadi H, Djafarian K. The effect of selenium therapy in critically ill patients: an umbrella review of systematic reviews and meta-analysis of randomized controlled trials. Eur J Med Res. 2023 Feb 28;28(1):104. doi: 10.1186/s40001-023-01075-w. PMID: 36849891; PMCID: PMC9972714. 50 3 RESPIRAČNÉ ZLYHANIE NA PODKLADE VÍRUSOVEJ INFEKCIE A JEHO LIEČBA V priebehu posledných pár rokov sa naplno preukázal deštrukčný potenciál vírusových pneumónii, pôsobiaci na pľúcne tkanivo. Okrem pochopenia zápalových mechanizmov vedúcich k poškodeniu alveolárnych jednotiek sa výrazne zdokonaľovali možnosti umelej pľúcnej ventilácie, ktorých cieľom bolo zlepšenie prežívania pacientov v akútnej fáze ochorenia, ako aj redukcia postinfekčných komplikácií. 3.1 Viachladinová ventilácia pľúc Umelá pľúcna ventilácia je základným postupom orgánovej podpory pacientov, u ktorých došlo k vzniku závažnej poruchy ventilačnej alebo oxygenačnej funkcie respiračného systému alebo sú takouto poruchou aktuálne ohrození. Medzi tieto stavy patrí syndróm akútnej respiračnej tiesne (ARDS), kardiogénny a nekardiogénny pľúcny edém, kontúzie pľúc, závažné pneumónie a ďalšie kritické stavy. Terapia závažnej respiračnej insuficiencie vyžaduje zabezpečenie dýchacích ciest a realizáciu umelej pľúcnej ventilácie s voľbou adekvátneho ventilačného režimu s individuálnym nastavením ventilačných parametrov vzhľadom na vlastnosti respiračného systému daného pacienta. Vulnerabilné pľúcne tkanivo, ktoré je poškodené už primárnym inzultom, je v priebehu liečby ďalej ohrozené samotnou umelou pľúcnou ventiláciou. Nepriaznivo na pľúcne tkanivo pôsobia vysoké inspiračné tlaky, ktorých následkom môže byť barotrauma, ako aj kaskádová aktivácia autoimunitných procesov, ktorých výsledkom je biotrauma pľúc. Pod pojmom biotrauma pľúc rozumieme preukázaný vplyv agresívnej ventilácie (vysoké špičkové inspiračné tlaky, extrémny vplyv strižných síl pri otváraní alveol a podobne), ktoré participujú na rozvoji lokálnej zápalovej odpovede v pľúcach, následkom čoho dochádza ku excesívnej tvorbe reaktívnych molekúl a tvorbe proinflamatórnych cytokínov. Tieto závery vychádzajú z viacerých klinických štúdií, v ktorých bola porovnávaná vysokotlaková ventilácia s „protektívnym" spôsobom ventilácie. Ranieri M a spol. zistili, že „tradičná UVP" (Vt 11 ml*kg-1, PEEP 6.7 cm H20) u pacientov s ARDS je spojená s významným nárastom pľúcnej a systémovej koncentrácie proinflamatórnych cytokínov (TNF-a, IL-lb, JL-6, IL-8) v porovnaní s „protektívnym" spôsobom ventilácie (Vt 7.6 mPkg"1, PEEP 14.8 cm H2O). Plötz F. a spol. 51 zistili, že ventilačný režim (Vr 10 mPkg"1, PEEP 4 cm H2O) už po dvoch hodinách aplikácie vedie k nárastu TNF-a a LL-6 v bronchoalveolárnej laváži u detí bez predchádzajúceho pľúcneho poškodenia. Tieto nálezy potvrdili existenciu VILI (ventilator-induced lung injury) v humánnej medicíne. Patogenetický mechanizmus biotraumy predpokladá mechanické pôsobenie umelej ventilácie na pľúca (vysoké špičkové tlaky, over-distenzia), energie dodávanej pri UVP ventilátorom, ktorá je pohltená pľúcami a aplikácie vysokých koncentrácii inšpirovaného kyslíka. Tieto vplyvy vedú k funkčným zmenám buniek v pľúcnom tkanive (up-regulation) a následnému vzniku zápalovej reakcie. Zápalová odpoveď sa cytologický prejavuje infiltráciou tkanivových štruktúr pľúc a bronchoalveolárnej tekutiny neutrofilmi (23 - 26). Zápalová reakcia indukovaná mechanickými inzultmi v pľúcach môže zhoršovať už existujúci infekčný zápalový proces v samotných pľúcach. Cestou produkcie zápalových mediátorov môže ďalej podporiť rozširovanie zápalovej reakcie, ako aj apoptotických biochemických signálov na sekundárne orgány. Pri ARDS a ťažkých pneumóniách je prítomné závažné poškodenie pľúcneho parenchýmu s redukciou alveolárnej plochy, čoho následkom dochádza k narušeniu výmeny plynov medzi vonkajším a vnútorným prostredím. Jedným z vážnych a ťažko riešiteľných problémov je nehomogénne poškodenie pľúcneho tkaniva, ktoré zapríčiňuje nehomogénnu distribúciu plynov. Rozdiely v časových konštantách (x) jednotlivých kompartmentov v pľúcach sú natoľko evidentné, že pri optimálnom nastavení parametrov UVP přejeden či dva kompartmenty, je nastavenie parametrov (frekvencia, pomer dôb Ti : Te, prietoky plynov) pre ďalšie pľúcne kompartmenty nevyhovujúce, alebo výrazne suboptimálne. Praktickým riešením je zavedenie viachladinovej (multilevel) ventilácie. Doterajšie experimentálne a klinické poznatky poukazujú na terapeutickú efektivitu viachladinovej ventilácie, ktorou je dosiahnutá redistribúcia alebo zlepšená distribúcia plynov z kompartmentov s krátkou časovou konštantou do oblastí s dlhšou časovou konštantou (24, 26 - 27). V rámci niekoľkých pilotných projektov bol náš vedecko-lekársky tím na Klinike anestéziológie a intenzívnej medicíny vo Východoslovenskom ústave srdcových a cievnych chorôb v Košiciach, zapojený do klinických štúdií, ktorých úlohou bola aplikácia matematických ventilačných modelov do klinickej praxe pri troj-stvor hladinovej ventilácii u pacientov s nehomogénnym postihnutím pľúc. Predmetom výskumu bola ventilácia pľúc pri nehomogénnom pľúcnom poškodení a jeho prevencia a neinvazívna diagnostika. Pri týchto viachladinových ventilačných režimoch základnú ventilačnú hladinu predstavoval PS alebo PC ventilačný režim s nastavenými parametrami a to: frekvenciou dýchania, inspiračných 52 tlakov, pomeru Ti: Te, PEEP. Nad základnou hladinou boli na pozadí nastavené nadstavbové ventilačné režimy, a to pri trojhladinovej (3-level) ventilácii PEEPhl a frekvencii PEEPhl. Pri štvorhladinovej (4-level) ventilácii boli nastavenia ventilátora doplnené o ďalšie parametre a to PEEPh2 a frekvencia PEEPh2. Frekvencie nadstavbových hladín ako aj aplikovaný tlak boli nižšie ako parametre základnej hladiny. 3.2 COVID - 19 V roku 2019 moderný svet začala ohrozovať globálna pandémia ochorenia COVID-19, ktorá bola spôsobená prenosom akútneho infekčného ochorenia vyvolaného koronavírusom SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2). Aj keď COVID-19 v tom čase vykazoval nižšiu mortalitu (0,25 až 3 %) v porovnaní so SARS (10 %), MERS (34 %) alebo H1N1 (17 %). Intenzita jeho šírenia v konečnom dôsledku viedla k vysokému počtu úmrtí. V niektorých európskych krajinách dosiahla mortalita na SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) 7-10 % z celkového počtu nakazených, čo predstavovalo 10 násobne vyššiu úmrtnosť v porovnaní s infekciou chrípky. Mortalita ochorenia COVID-19 vzhľadom na ľahšie šírenie ochorenia a postihnuté vysoké percento populácie bola v absolútnych číslach vysoká (28). Ochorenie COVID-19 vedie k rozvoju celého spektra respiračných následkov s extrémne vysokou incidenciou pneumónie a syndrómu akútnej respiračnej tiesne (29). Na Slovensku sa prvý prípad objavil 6. marca 2020. V rámci svetovej iniciatívy hľadania efektívnych preventívno-liečebných postupov bol náš vedecko-lekársky tím zapojený do grantových projektov podporovaných EU zameraných na zlepšenie programov ventilácie nehomogénnych pľúc. Grantové projekty: 1. Operačný program Integrovaná infrastruktura pre projekt „Návrh a implementácia pokročilých metód ventilačnej liečby a diagnostiky vírusových pneumónií vrátane COVID-19 s možnosťou ich rýchleho osvojenia", kód 313011ASX1, akronym IPMVDCov, spolufinancovaný zo zdrojov Európskeho fondu regionálneho rozvoja v spolupráci s Ministerstvom školstva, výskumu, vývoja a mládeže Slovenskej republiky. 53 2. Operačný program Integrovaná infrastruktura pre projekt „Výskum a vývoj systému zefektívnenia ventilácie pacientov s COVID-19 alebo iným nehomogénnym poškodením pľúc" kód 313011ATG9 spolufinancovaný zo zdrojov Európskeho fondu regionálneho rozvoja v spolupráci s Ministerstvom školstva, výskumu, vývoja a mládeže Slovenskej republiky. 3.3 Práce súvisiace s grantovými projektanti Práce súvisiace s grantovými projektmi boli publikované v časopise Anestéziológia a intenzívna medicína ISSN 1339-4177. • Rybár D, Depta F, Kočan L, Grendel T, Nosál'N, Imrecze S, Tôrôk P. Hélium a jeho možné využitie v intenzívnej medicíne. Anestéziológia a intenzívna medicína. 2023; 12(2):50-54. • Tôrôk P, Rybár D, Depta F, Nosál'M, Kočan L, Donič V, Grendel T, Firment P, Imrecze S, Firment J. F 0.1 (Flow in ld.00 msec.) ako parameter ventilačného úsilia počas ventilačnej podpory. Anestéziológia a intenzívna medicína. 2023; 12(2):55-57. • Tôrôk P, Rybár D, Depta F, Nosál'M, Kočan L, Donič V Grendel T, PhD., MUDr. Firment P, Imrecze S, Firment J. Nehomogénne pľúcne poškodenie - teória vplyvu časového faktora na výmenu plynov v pľúcach. Anestéziológia a intenzívna medicína. 2023;12(1):11-15. • Tôrôk P, Depta F, Nosál'M, Kočan L, Donič V, Grendel T, Rybár D, Imrecze S, Firment J. Základné fyzikálne, matematické a biologické princípy očisťovania dýchacích ciest pri umelej ventilácii pľúc. Anestéziológia a intenzívna medicína. 2022;ll(2):51-55. • Nosál'M, Kočan L, Tôrôk P, Donič V, Grendel T, Rybár D, Depta F, Firment P, Imrecze S. Fyziológia apatofyziológia problémov spojených s dlhodobou mechanickou ventiláciou pľúc. Anestéziológia a intenzívna medicína. 2022;11(1):11-14. 54 • Kočan L, Vašková J, Tôrôk P, Donič V, Grendel T, Nosál'M, Rybár D, Depta F, Firment P, Imrecze S. Terapeutické možnosti vodíka pri vybraných patologických stavoch u kriticky chorých pacientov. Anestéziológia a intenzívna medicína. 2022;11(1):15-19. Za doterajšie výsledky súvisiace s menovanými grantovými projektmi bola udelená nášmu lekársko-vedeckému tímu CENA ZA TRANSFER TECHNOLÓGIÍ NA SLOVENSKU 2023 v kategórii INOVÁCIA. Národným portálom pre transfer technológií Názov projektu: Zariadenie na umelú ventiláciu pľúc s identifikáciou nehomogenity distribúcie plynov a spôsob riadenia zariadenia pri umelej ventilácii pľúc. Majiteľ technológie: Univerzita Pavla Jozefa Šafárika v Košiciach (Príloha č.2). Pôvodcovia: doc. MUDr. Pavol Tôrôk, CSc, prof. MUDr. Viliam Donič, PhD. , MUDr. Tomáš Grendel, PhD., MUDr. Ladislav Kočan PhD., MUDr. Martin Nosál', MUDr. Dušan Rybár, PhD., MUDr. Filip Depta, MUDr. Štefan Imrecze PhD., doc. MUDr. Jozef Firment, PhD., MUDr. JUDr. Peter Firment 3.4 Oxidačný stres pri vírusovej pneumónii Experimentálne štúdie na zvieracích modeloch zameraných na výskum priebehu akútneho respiračného syndrómu (ARDS) pri vírusových infekciách potvrdili prítomnosť excesívnej zápalovej reakcie v pľúcnom tkanive. V priebehu týchto zložitých interakcií zohráva dôležitú úlohu vrodená imunita a aktivácia dráh súvisiacich s progresiou oxidačného stresu. Úloha polymorfonukleárov v tvorbe ROS je dlhodobo známa. Zložité interakcie vedú k aktivácii NF4d3 v imunukompetentných bunkách, a to indukciou zápalu a aktiváciou signálnych dráh hostiteľskej bunky sprostredkovaných proteinkinázami p38 MAPK a zapojením neštruktúrnych 3a proteínov, kódovaných vírusovým genómom SARS-CoV a ďalších procesov, ktoré vedú k zvýšenej indukcii bunkovej apoptózy. Ďalšie publikované práce Lin a kol. poukazujú na súvislosť medzi vírusovou infekciou a aktiváciou excesívnej tvorby ROS vo vývojovej rade bielych krviniek, promonocytov FLL-CZ bunkách. Ich aktivácia je indikovaná vírusovou proteázou 3CLPro (3-chymotrypsin like protease). Tieto zložité interakcie môžu byť základom progresie vážneho poškodenia pľúc u 55 infikovaných jedincov, poháňaného aktiváciou dráh oxidačného stresu zložito spojených s vrodenou imunitou. Okrem zápalovej reakcie spustenej vírusovou infekciou, zvýšený prooxidačný stav v pľúcnom tkanive dopĺňa možná biotrauma pľúc spôsobená energiou prenesenou z ventilátora na tkanivá pľúc, ďalej inšpirácia vysokých frakcií kyslíka ako aj celkový septický stav pacienta (30, 31). 3.5 Postpneumonická fibróza pľúc Pľúcna fibróza je chronickou komplikáciou závažnej vírusovej pneumónie, častokrát sprevádzanej ARDS. Vo všeobecnosti sú pľúca veľmi fragilné voči rôznym noxám. Zraniteľnosť vychádza zo samotnej štruktúry pľúcneho tkaniva, ktorou je nízka hustotu buniek v pomere k objemu pľúc (32). Intersticiálne pľúcne abnormality vznikajúce v nadväznosti na ťažké postihnutie pľúc rôznej etiológie sú známou komplikáciou už z éry pred pandémiou COVLD-19. Nálezy fibrotických zmien na pľúcach boli popísané u pacientov s potvrdenou infekciou SARS-CoV Severe-Acute-Respiratory-Syndrome coronavirus) alebo MERS-CoV (Middle-East-Respiratory-Syndrome coronavirus). Vo väčšine štúdií zaoberajúcich sa problematikou rozvoja post-vírusovej pľúcnej fibrózy je cytokínová búrka považovaná za primárny patogenetický mechanizmus. Celý tento stav výrazne urýchľuje prítomný oxidačný stres, pri ktorom dochádza ku amplifikácii kaskádových dejov a k ďalšiemu masívnemu uvoľňovaniu prozápalových cytokínov v rámci aberantnej zápalovej reakcie. Rozvoju pľúcnej fibrózy ďalej napomáha dysregulácia reparačných dejov (32, 33). Vírusové infekcie, medzi ktoré patrí COVLD-19, vyvolávajú zmeny v bunkovom a molekulárnom prostredí pľúcneho tkaniva, čím spúšťajú nadmernú expresiu zápalových cytokínov, najmä transformujúceho rastového faktora-pi (TGF-P), tumor nekrotizujúceho faktora-a (TNF-a) interleukín-1 (LL-1) a interleukín-6 (LL-6). Tieto mediátory stimulujú proliferáciu alveolárnych buniek typu 2 a nábor fibroblastov, čo vyvrcholí zvýšenou produkciou a ukladaním extracelulárnej matrix (ECM). Tento proces ohrozuje architektúru alveolárno-kapilárnej membrány, čo vedie k zhoršenej výmene plynov a hypoxémii. Alveolárna regenerácia po akútnom poškodení pľúc bola pozorovaná v rámci zvieracích štúdií, v ktorých sa preukázala kľúčová funkcia pneumocytov II typu (AT2). AT2 bunky ako základné štrukturálne zložky alveolárneho epitelu sú schopné sa ďalej proliferovať a diferencovať sa na pneumocyty I typu (ATI). ATI bunky vytvárajú funkčnú štruktúru alveolov (32, 34, 35). 56 Vírus SARS-CoV-2 môže priamo infikovať AT2 bunky a spôsobiť ich masívnu apopotózu. Dôležitú úlohu v tomto procese zohrávajú zápalové mechanizmy. Nieje známe, či alveolárna regenerácia prebieha rovnakým spôsobom u ľudí po poškodení pľúc vyvolanom infekciou SARS-CoV-2 ako na zvieracích modeloch. Rovnako otázny je časový interval regenerácie. Pri dlhotrvajúcom a deštruktívnom poškodení pľúc dochádza v miestach poškodenia k tvorbe a hromadeniu väzivových vlákien. Jedná sa o reparačný mechanizmus, pri ktorom sa vytvára väzivové tkanivo. Charakteristická je kumulácia väziva v mieste poškodenia epitelu a endotelu s významnou redukciou funkcie pľúc a zvýšenou morbiditou. Pľúcna fibróza je bežným výsledkom väčšiny chronických zápalových pľúcnych porúch a môže mať vplyv na funkcie pľúc, čo v konečnom dôsledku vedie k respiračnému zlyhaniu a smrti.. Proces tvorby väziva závisí aj od závažnosti a trvania poškodenia, keďže dlhotrvajúce poškodenia majú tendenciu rozvinúť sa na fibrózu na rozdiel od poškodení malého rozsahu. Zistilo sa, že interakcie medzi rôznymi typmi buniek sú veľmi dôležité pre nástup fibrózy (36). Za kľúčové v tomto procese sa považujú mezenchymálne bunky a fibroblasty. Ďalšími dôležitými procesmi sú aktivácia glykolýzy vo fibroblastoch po poškodení pľúc a kaskáda enzymatických aktivácií, ktoré zvyšujú proliferáciu buniek, syntézu kolagenu a produkciu sekundárnych metabolitov, čím podporujú fibrózu. Pri aktivácii fibroblastov hrá dôležitú úlohu zvýšená glutaminolýza a oxidácia mastných kyselín. Zároveň rezidentně monocyty a makrofágy majú regulačnú úlohu v procesoch tkanivovej fibrózy. Pomáhajú iniciovať, udržiavať a upravovať poškodenie tkaniva (35, 36). Úroveň zápalovej reakcie je rozhodujúcim faktorom či dôjde k regenerácii alebo fibróze pľúcneho tkaniva. Oba procesy náhrady poškodeného tkaniva sú často sekvenčné a vzájomne prepojené. V prípade obmedzeného poškodenia sa regenerácia zvyčajne vyskytuje prioritne, aby sa obnovila integrita a funkcia tkaniva. Ak tento proces zlyhá v dôsledku vážneho poškodenia iniciuje sa tvorba väziva, ktorá môže viesť k chronickému ochoreniu pľúc alebo kolapsu. To naznačuje, že regenerácia je plnohodnotný proces, zatiaľ čo tvorba väziva je dobrá len vtedy, keď je mierna. V prípade vírusovej infekcie je zápalové poškodenie výrazné a fibróza je dominantným procesom v porovnaní s regeneráciou (34, 35). Otázkou je reverzibilita započatej fibrogenézy jej možná prevencia, ako aj následná rekonvalescencia pacientov a kvalita života vrátane trvalých positnfekčných následkov. Táto téma je aktuálna vzhľadom na veľké percento pacientov po prekonanej COVLD infekcii a ďalšej predikcie ich prognózy. Prvé skúsenosti so závažnou vírusovou pneumóniou a problematickou ventiláciou ťažko postihnutých pľúc sme získali na našom pracovisku počas epidémie prasacej chrípky H1N1. Do sledovanej skupiny pacientov v septickom šoku v rámci 57 štúdie Se-AOX bol v roku 2012 zaradený 30 ročný pacient so závažným priebehom vírusovej pneumónie H1N1. U pacienta bola prvýkrát použitá viachladinová ventilácia vrátane intravenóznej suplementácie selénu a glutamínu. Pacient po 60-tich dňoch opúšťa nemocnicu v zlepšenom stave avšak s ťažko postihnutými pľúcami do domácej opatery. Pacient bol ďalej sledovaný počas nasledujúcich 10 rokov, s realizovanými opakovanými RTG vyšetreniami pľúc, ktoré poukazovali na postupné zlepšenie stavu pľúcneho parenchýmu. V roku 2022 pacient tragicky zahynul a telo bolo odoslané na anatomicko-patologicku pitvu. Závery z histologických nálezov pľúcneho tkaniva poukazujú na úplnú reparáciu poškodeného tkaniva. Táto kazuistika bola publikovaná v karentovom medicínskom časopise MEDICÍNE Baltimore ISSN: 1536-5964 Q3, Impact Factor: 1.6. 58 I Clinical Case Report Medicine Full recovery of lung tissue after severe viral pneumonia H1N1 A case report with 10 years follow-up Ladislav Kočan, MD, PhL>, Jozef Firment, MD, PhDb, Ingrid Pirníková, MDD, Silvia Farkašová lannaccone, MD, PhDc. Dušan Rybár, MD, PhD3, Juliana Gnoriková, MDd, Ján Korček, MDb, Hana Kočanová, MDe, Pavol Tôrók, MD, PhDa Simona Rapčanová, MDf, Janka Vašková, Dr, PhD9*# Abstract x Rationaie: World healthcare frequently faced severe viral pneumonia cases in the last decades, due to pandemic situations such as H1N1, MERS-CoV, and SARS-COVID-19. Patient concerns: The impact of viral infection on lung structure, lung function, and overall mortality was significant. The quality of life and assumed life expectancy was decreased with the supposed development of lung fibrosis in involved sun/ived patients. Diagnoses: We described the course and treatment of severe pneumonia H1N1 in a 30-year-old patient. Interventions: Patient was included in a study regarding the therapeutic efficacy of selenium ClinicalTrials.gov ID: NCT02026856 with 10 years follow-up with concurrently documented X-ray lung examinations and final histology of lung tissue after sudden death. Outcomes: All sequential examinations and histological findings show a healing trend with the final full recovery of lung tissue. Abbreviations: AT1 - alveolar type I cell, AT2 = alveolar type II cell, GT = computed tomography, H1N1 = type of influenza A virus, swine flu, PEEP = positive end-expiratory pressure, SARS-Cov-2 = severe acure respiratory syndrome-related Coronavirus 2. Keywords: covid-19, Hf N1, lung fibrosis, pneumonia, recovery, viral infection 1. Introduction Pneumonia causes high morbidity and mortality with high hospitalization rate worldwide. Viral pneumonias are characterized by epidemic and pandemic spread in certain time cycles. Examples are the Spanish flu (1918-1920) with an estimated 500 million infected, 50 to 100 million victims, the Asian flu (1957-1958) responsible for 1 to 1.5 million deaths, or the Hong Kong flu, which broke out in in 1969, with an estimated 750 million infected patients and rip to 1 million victims.1'1 In the interpan-demic period, viral pneumonias are considered less serious compared to bacterial pneumonias. However, mortality from viral lung infections has increased dramatically in the last decade with the emergence of new strains of viruses such as the pandemic A type of influenza A virus, swine flu (H1N1) 2009 virus, which is a combination of swine, avian and human influenza viruses, and as well as the emergence of a new strain of the Middle East respiratory syndrome Coronavirus in the Middle East.'2' Since 2019, the human society has faced a new pandemic threat caused by the severe acure respiratory syndrome-related Coronavirus 2 (SARS-CoV-2) virus, in various evolving variants. The manifestation of the infection is the disease COVID-19 with the dominance of respiratory symptoms, including viral pneumonia.1*1 The treatment strategy of mild respiratory forms of COVID-19 to severe clinical conditions associated with respiratory This publication is the result of the project implementation: Design and implementation of advanced methods of lungs ventilation treatment and diagnosis of viral pneumonia, inducting Covid-19 with the possibility of their rapid adoption", ITMS20Í4+: 313011ASX1 supported by the Operational Programme Integrated Infrastructure, funded by the ERDF. Informed consent about study enrollment and follow up was obtained from participant included in the study during the first hospital admission in 2011. The authors have no conflicts of interest to disclose. The datasets generated during and/or analyzed during the current study are not publicly available, but are available from the corresponding author on reasonable request "Clinic of Anaesthesiology and Intensive Care Medicine, East Slovak Institute of Cardiovascular Disease, Košice, Slovak Republic," 1st Clinic of Anestesiology and Intensive Medicine, Faculty of Medicine, Pavol Jozef Šafárik University in Košice and Louis Pasteur University Hospital, Košice, Slovak Republic, ~ Department of Forensic Medicine, Faculty of Medicine, Pavol Jozef Šafárik University In Košice, Košice, Slovak fíepubíic, c Department of Anaesthesiology and Intensive Care Medicine, Štefan Kukura Hospital with Policlinic, Michalovce, Slovak Republic, ŕ Clinic of Anaesthesiology and Intensive Care Medicine, Railway Hospital and Clinic Košíce, Slovak Republic,' Europainctinics, Bardejov, Slovak Republic, s Department of Medical and Clinical Biochemistry. Faculty of Medicine, Pavol Jozef Šafárik University in Košice, Košice, Slovak Republic. * Correspondence: Janka Vašková, Department of Medical and Clinical Biochemistry, Faculty of Medicine, Pavol Jozef Šafárik University in Košice, Tr. SNP 1, Košice 04066, Slovak Republic (e-mail: janka.vaskova@upjs.sk). Copyright © 2023 the Auihor(s). Published by Welters Kluwer Health, inc. This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work Is properly cited. How to cite this article: Kočan L, Firment J, Pirníková I, Farkašová lannaccone Sr Rybár D, Gnoriková J, Korček J, Kočanová H, Tôrôk P, Rapčanová S, Vašková J. Full recovery of lung tissue after severe viral pneumonia H1N1:A case report with 10 years follow-up. Medicine 2023; 102;8(e33052). Received: 24 January 2023 /Accepted: 1 February 2023 http://dx.doi.org/10.1097/MD.0000000000033052 59 Kočanetal, • Medicine (2023} 102:8 Medicine failure with the development of the acute respiratory distress syndrome and associated complications has brought a spectrum of new therapeutic procedures. At the same time, new data on the health status or patients after overcoming more severe forms of COV1D-19 point to a new problem, which is a kind of "hidden pandemic" of post-COVID consequences. The conclusions of observational clinical studies show that up to 90% of patients hospitalized with COVID-19 have symptoms of dyspnea, reduced diffusion capacity of the lungs, related to lung tissue damage., and subsequently a decrease in physical activity after infection. In more than 50% of patients symptoms disappear within 3 months, while it is assumed that these patients are going to have a complete regeneration of lung tissue within 9 months after the end of hospitalization. A significant part of the population has some pre-disease lung tissue damage already, and almost 10% of all patients show nbrotic lung damage even before the infection of COVID-19. Predisposed patients are exposed to a greater risk of developing post-COVID nbrotic remodeling of the lung parenchyma. The 2% to 6% incidence of post-COVID pulmonary fibrosis can he estimated after moderate respiratory involvement. The rate of lung remodeling increases with the severity of the disease course.'41 However, not all clinically serious pneumonias caused by viruses lead immediately to fibrosis. The case report shows a retrospective view of the regenerative processes of the patient's lung parenchyma after an H1N1 infection with acute respiratory distress syndrome in the acute phase of the disease, his physical lung computed tomography (CT) and X-ray findings during the next 10 years of follow-up, and histological findings of lung tissue from the patient's autopsy. 2. Case report A 30-year-old patient hospitalized in January 2011 at the Intensive Care Unit of University Hospital due to bilateral pneumonia presented with respiratory failure with an oxygenation index < 200. At admission, intubation and initiation of eon-trolled ventilation was started. Pressure controlled ventilation with fraction of inspired oxygen of 0.8, positive end-expiratory pressure (PEEP) 9 cm H30, pressure controlled ventilation 22cm H,0 and minute ventilation 12 l/minute was applied along with sedation, continuous administration of muscle relaxants, and prone positioning. Vasopressor support with a norepinephrine dose of up to 0.15 ug/kg/minute was necessary to stabilize circulation. Empirical antibiotic treatment and anticoagulation therapy was started (Fig. 1). Viral infection of influenza A type H1N1 was diagnosed by the PCR method, thus a virostatic agent Oseltamivir was added. Restrictive fluid administration was maintained daily. Antifungal agent Voriconazol was later added for the positive galactomannan test. The patient's condition deteriorated as he developed severe sepsis. Pseudomonas aeruginosa was confirmed from the bronchoalveolar lavage leading to the adjustment of antibiotic treatment (Fig. 1) and adding immu-nomodulation therapy with intravenous immunoglobulin and Polyoxidonium. On the 5th day the so called programmed multilevel ventilation mode performed by the Chirana AURA V (Slovakia) ventilator was applied for the first time with basic PEEP level 7 to 9 cm H20, pressure control 12 to 10 cm H20, frequency of 22 to 23/minute, upper PEEP level of 5 to 6 cm H,0 was applied 4 to 5 times per minute, resulting in minute ventilation of 12 to 8 l/minute. Cortisol therapy started from the 5th day of hospitalization in a daily dose of 200 mg, later reduced to lOOmg, followed by reduction to 50mg per day during the overall 15-day corticosteroid therapy. On 5th day, selenium adjuvant therapy started too. Patient was enrolled in the study Se-AOX approved by the ethical committee of Pavol Jozef Šafárik University in Košice under number 109/2011; ClinicalTriaIs.gov identifier: NCT02026856. Selenium in the form of sodium selenite pentahydrate at 750mg/day for 6 days as a continual infusion was supplemented (lOOOmg of sodium selenite pentahydrate = 333 fig of selenium) (Selenasc, Vivax). During the severe sepsis, renal insufficiency developed with oliguria and an increase in blood levels of the urea and creatinine which necessitated the initiation of continuous veno-venous hemodialysis. Diarrhea developed 11 days from admission. In the next course, the patient's lung compliance and resistance started to improve. A gradual ventilation weaning was started by switching the 3-level ventilation to pressure control and later to pressure support ventilation mode with Pps 10 to 6cm H20, PEEP 7 to 5cm H20 and minute ventilation 10-7 1/ minute. On the 14th day a CT scan of the lungs showed bilaterally present diffuse opacities of the milk glass type with thickening of the interlobular septation (Fig. 2). Pe r i b ion ch o v a sc u I a r irreguíar consolidations were present in the upper and posterobasal lung segments of the lower lung lobes with a negative bronchogram. In superposition with the described changes, there was a suspicion of micro- and macrocystic remodeling of the lung parenchyma (so called honeycombing) on the periphery of rhe left lingular lung segments and the right medial segment of the middle lung lobe. The pleural spaces were free from fluid effusions. On the 15th day of hospitalization, percutaneous dilatation tracheostomy was performed. 17 days after admission, clipping was attempted for gastrointestinal bleeding from a duodenal ulcer- unsuccessfully, therefore, and a laparotomy with successful duodenotomy was performed in the D2 to 3 region. In the postoperative period, paralytic ileus developed and was treated conservatively. Subsequently, enteral nutrition was started once again. In the next course of hospitalization, repeated attempts to wean the patient from controlled ventilation were made. Concurrently, withdrawal symptoms appeared after long-term application of opioids, therefore, and neuroleptics were started. The patient gradually improved and was able to breathe spontaneously for several hours a day through aT-tube with intermittent pressure support ventilation. Continuous renal replacement therapy was switched to intermittent hemodialysis. Rifaximin Cefepime Tobramycine Linezolid Meropenem Metronidazol Metronidazol Vancomycins Imipenem Meropenem Vorikonazol Linezolid Ceioper&z one Oseltamiv r 1st_5th_10th_15th_20th_25th_30th_35th_40th Figure 1. Time sequence of dynamic changes in antimicrobial treatment according to empirical experiences of the intensive Care Unit, prospectively medication followed according to microbial culture and its sensitivity, and individual patient toleration to drugs. 2 60 Kocan et ai. • Medicine (2023) 102:8 www.md-journal.corn Figure 2. CT sagittal and a coronal view of lungs. In the upper row, the red cross marker is focused on the left lung. In lower row, the red cross marker is focused on the right lung. CT = computed tomography. After repeated negative cultures from biological samples, antibiotic treatment was terminated. At that time, the patient was on ah oral full-fledged diet and adhered to a complex rehabilitation process. In the following days, the patient developed a polyuric phase of acute kidney injury followed by a gradual recovery of renal functions. On the 47* day, the patient was transferred to the intensive care unit of the regional hospital. The patient further improved and was discharged to outpatient care. In 2012 and 2014 the patient was treated by the emergency medical service for difficult breathing leading to admission to the local hospital's internal medicine department. In April 2018, the patient was hospitalized in the local hospital's intensive care unit due to acute pancreatitis as a result of excessive alcohol consumption, confirmed by laboratory and CT examination. The condition was complicated by the development of disorientation, agitation, and delirium, which necessitated the use of sedatives and antipsychotics. Despite the treatment, the condition did not improve,, furthermore, and respiratory insufficiency developed. The patient was intubated and controlled ventilation was started and vasopressors were temporarily used. The following day, the symptoms improved, sedation was gradually discontinued, ventilation support was reduced and finally the patient was extubated. He was transferred to the surgical department. The control X-ray of the chest from this period describes findings without serious pathology, presenting indistinct irregularities of the course bronchovascular pattern in the lower lung fields (Fig. 3). The patient was discharged home after 7 days. Control X-ray examination in following 2-years period shows Figure 4. In November 2021, the patient was transported by the emergency medical service to the local hospital's emergency department after falling on the street. Coincidentally, a diagnosis of COVID-19 by antigen testing was made. The patient was examined by a traumatologist, a neurologist, and an internist. A small subarachnoid hemorrhage was found on the left temporal side on the head CT scan. Laboratory results showed a significant elevation of hepatic enzymes, leukopenia, thrombocytopenia, moderate elevation of C-reactive protein, D-dimers, and fibrinogen. Abdominal ultrasound revealed cirrhosis of the liver. The patient had repeated grand mal seizures and developed delirium. In the evening, a cardiac arrest suddenly occurred. Immediately, advanced cardiopulmonary resuscitation was started but was unsuccessful and the patient died. An autopsy was indicated. Autopsy was performed at the Medico-Legal and Pathological-Anatomical Department of Health Care Surveillance Authority in Kosice within 24 hours after death. Death was attributed to severe cerebral edema following traumatic brain injury (subarachnoid hemorrhage, focal contusions of the brain). The right and left lungs weighted 535g and 515 g respectively. The pleural surfaces were smooth with a mild accumulation of anthracotic pigment deposited along lymphatic routes in the pleura (anthracosis). The upper lobes demonstrated dilatation of air spaces; the lower lobes were dry, firm, airless and consolidated. The parenchyma of the lower lobes was pink-red to red-purple in color with a meaty appearance and showed no expression of fluid from the cut surfaces upon compression (Fig. 5). The bronchi were white-gray with no sign of fluid collection. Histological examination of hematoxylin-cosin stained sections taken from both lungs 3 Kocan et ai. • Medicine (2023) 102:8 www.md-journal.com Figure 5. A: Lungs: the upper lobes with dilatation of air spaces; the lower lobes were dry, firm, airless and consolidated. B: Lungs: acute congestion, focal hemorrhage, focal emphysema, mild alveolar edema, scant interstitial lymphoplasmacytic infiltrate, some interstitial fibrosis (hematoxylin and eosin, x40). C: Lungs: acute congestion, hemorrhage, mild aiveoiar edema; foca! emphysema (hematoxylin and eosin, x1Q0). □: Lungs: acute congestion, focal hemorrhage, focal emphysema, mild alveolar edema, scant interstitial lymphoplasmacytic infiltrate, some interstitial fibrosis (hematoxylin and eosin, x40), patients indicate that signs of pulmonary fibrosis may subside over time.14' The mentioned case shows the high regenerative capacity of the lungs after overcoming severe viral pneumonia, white the initial prognostic assumption was the fibrotic remodeling of the lung tissue. This topic is relevant due to the large percentage of patients who have overcome the COV1D infection and the further prediction of their prognosis. In general, lungs are very fragile against various noxae. Vulnerability stems from the very structure of the lung tissue, which is the low density of cells in relation to the lung volume. Lung function depends on the arrangement of cells forming alveolar septa.1'1 The entry gate of the SARS Cov-2 virus into the cells of the respiratory system is the membrane receptor for angio-tensm-converting enzyme 2 or the membrane protein transmembrane serine protease 2. The virus is able to infect alveolar type I cells (ATI) and alveolar type 11 cells (at2). It is believed that only a small population of at2 cells express the angioten sin-con verting enzyme 2 receptor on their surface.|S| The most vulnerable cells are located on the surface of the alveoli.171 Damage to ATI stimulates the rapid proliferation and differentiation of at2 cells, which also regenerates the tissue barrier function.151 Alveolar regeneration after acute lung injury has been observed in animal studies demonstrating the key function of at2 cells. at2 cells, as basic structural components of the alveolar epithelium, are able to further proliferate and differentiate into ATI cells. ATI cells form the functional structure of the alveoli. However, evidence of lung tissue regeneration in humans is absent, probably due to the lack of lung samples obtained. The SARS-CoV-2 virus can directly infect at2 pneumocytes and cause their massive apophysis. Inflammatory mechanisms play an important role in this process. It is not known whether alveolar regeneration occurs in the same way in humans after lung injury induced by SARS-CoV-2 infection as in animal models. The time inrerval of regeneration is also questionable. In rodent animal models, lung regeneration and recovery of function takes several weeks.m Studies dealing with the recovery of respiratory functions in humans point to processes that last several years, which implies a longer onset of differentiation of at2 cells, in contrast to animal studies on rodents, in which this process lasts only a few weeks.1*1 In case of long-lasting and destructive damage to the lungs, the formation and accumulation of fibrous connective tissue (fibrosis) occurs in the places of damage, it is a reparative mechanism in which fibrous tissue is formed. Accumulation of fibrous tissue at the site of damage to the epithelium and endothelium with a significant reduction in lung function and increased morbidity is characteristic. Pulmonary fibrosis is a common outcome of most chronic inflammatory lung disorders and can affect lung function, ultimately leading to respiratory failure and death.|yf The process of fibrous tissue formation also depends on the severity and duration of the injury, as long-lasting injuries tend to develop into fibrosis in contrast to small-scale injuries. Interactions between different cell types have been found to be very important for the onset of fibrosis.'*1 Mesenchymal cells and fibroblasts are considered to be key in this process. Other important processes are the activation of glycolysis in fibroblasts after lung injury and a cascade of enzymatic activations that increase cell proliferation, collagen synthesis, and production of secondary metabolites, thereby promoting fibrosis. Increased glutaminolysis and fatty acid oxidation play an important role in the activation of fibroblasts. At the same time, resident monocytes and macrophages have a regulatory role in tissue fibrosis processes. They help initiate, maintain, and repair tissue damage.f8-101 The level of the inflammatory response is a decisive factor whether regeneration or fibrosis occurs. Both processes of damaged tissue replacement are often sequential and interconnected. In the case of limited damage, regeneration usually occurs preferentially to restore tissue integrity and function. If this process fails due to severe damage, fibrous tissue formation is initiated, and which can lead to chronic lung disease or collapse. This 62 Kočan et al, • Medicine (2023} 102:8 Medicine suggests that regeneration is a full-fledged process, while fibrous tissue formation is only good when it is moderate. In the case of viral infection, and inflammatory damage is prominent and fibrosis is the dominant process before regeneration.'10'111 Factors such as the amount and types of cells that are damaged, the disruption of barrier function, the intensity and duration of the local immune response can be a predictor of whether the damaged lung regenerates ad integrum or the formation of fibrous tissue leads to chronic lung disease.151 Study Se-AOX, in which this patient was also included, pointed out, for example, to a significant improvement or respiratory functions quantified on the basts of Carrico index in early selenium adjuvant therapy.1121 4. Conclusion The case report of a patient affected by severe viral pneumonia points to an example of possible complete regeneration of lung tissue, despite severe lung damage. Pathological changes in cellular organization within reparations include the formation of fibrous connective tissue, which inevitably changes the critical structure of lung properties and leads to poorer lung function. However, clinical experience and several studies have shown that the respiratory system has an extensive capacity to respond to noxae by regenerating damaged cells or proliferating and differentiating progenitor cells or changing the function of already existing differentiated cells. Pathological studies show that in damaged lung tissue, AT2 cells are the most extensive proliferating population in severely damaged lungs. Alveolar regeneration can be initiated especially in patients with less severe viral pneumonia. Author contributions Conceptualization: Ladislav Kočan, Jozef Firment, Pavol Torok, Janka Vašková. Data curation: Ladislav Kočan, Jozef Firment, Ingrid Ptrniková, Silvia Farkašová lannaccone, Dušan Rybár, Juliana Gnoriková, Ján Korček, Pavol Tórok. Formal analysis: Ladislav Kočan, Ingrid Pirniková, Silvia Farkašová lannaccone, Dušan Rybár, Juliána Gnoriková, Ján Korček, Hana Kočanová, Pavol Torók, Simona Rapcanova, Janka Vašková. Investigation: Ladislav Kočan, Jozef Firment, Janka Vašková. Methodology: Ladislav Kočan, Juliána Gnoriková, Ján Korček. Supervision: Joz,ef Firment, Silvia Farkašová lannaccone. Validation: Ladislav KoČan, Jozef Firment, Ingríd Pírníková, Simona Rapcanova. Writing - original draft: Ladislav Kočan, Silvia Farkašová lannaccone, Dušan Rybár, Hana Kočanová, Pavol Török, Janka Vašková. Writing-review &c editing: Ladislav KoČan, Jozef Firment, Silvia Farkašová lannaccone, Hana Kočanová, Simona Rapcanova, Janka Vašková. References [1] Akin L, Gozel MG. Understanding dynamics of pandemics. Turk J Med Sci. 2020;50:515-9. [2] Al-Baadani AM, Elzein FE, Alhemyadi SA, et ai. Characteristics and outcome of viral pneumonia caused by influenza and Middle Easr respiratory syndrome-eoronavirus infections: a 4^year experience from a tertiary care center. Ann Thorac Med. 2019;14:179-85. [3] McDonald IX Healing after COVID-19: are survivors at risk for pulmonary fibrosis? Am J Physiol Lung Cell Mol Physiol. 2021 ;320:L25 7-65. [4] Kotton DN, Morrisey EE. Lung regeneration: mechanisms, applications and emerging stem cell populations. Nat Med. 2UI4;20:fi22-32. [5] Lucas A, Yasa J, Lucas M. Regeneration and repair in the healing lung. Clin Transl Immunology. 2020,9x-l 1 52. [6] Zou X, Chen K, Zou J, et al. Single-cell RNA-seq data analysis on the receptor ACE2 expression reveals the potential risk of different human organs vulnerable to 2019-nCoV infection. Front Med. 2020;14:185-92. [7] Zhao F, Ma Q, Yue Q, er al. SARS-CoV-2 infection and lung regeneration. Clin Microbiol Rev. 2022;35:e0018S21. [8] Chen J, Wu H, Yu Y, et al. Pulmonary alveolar regeneration in adult COVID-19 patients. Cell Res. 2020;3:708-10. [9] Kobayashi Y, Tata A, Konkimalk A; et al. Persistence of a regeneration-associated, transitional alveolar epithelial cell state in pulmonary fibrosis. Nat Cell Biol. 2020;22:934^,6. [10] Gupta SK, Srivastava M, Minocha R, et al. Alveolar regeneration in COVID-19 patients: a network perspective. Int J Mol Sci. 2021;22:11279. [11] Yu M, Liu Y, Xu D, et al. Prediction of the development of pulmonary fibrosis using serial thin-section CT and clinical features in patients discharged after treatment for COVID-19 pneumonia. Korean J Radiol. 2020;21:746-55. [12] Koc.m L, Vaskova J, Vasko L, et al. Selenium adjuvant therapy in septic patients selected according to carrico index. Clin Biochem. 2014;47:44-50. 6 4 ÚLOHA OXIDAČNÉHO STRESU V CHRONICKEJ BOLESTI Oxidačný stres sa podieľa na rozvoji mnohých patologických stavov vrátane syndrómov spojených s chronickou neuropatickou bolesťou. Neuropatická bolesť vzniká z komplexnej súhry viacerých mechanizmov vrátane periférnej a centrálnej senzitizácie, ektopickej aktivity v primárnych aferentných vláknach, zmien v regulácii iónových kanálov, ako aj nerovnováhy medzi excitačnou a inhibičnou intercelulárnou a extracelulárnou signalizáciou. Pribúdajúce dôkazy odhalili, že „zápal nervového systému (angl. neuroinflammation - ďalej len neurozápal)" a oxidačná dysfunkcia zohrávajú rozhodujúcu úlohu pri indukcii a udržiavaní neuropatickej bolesti. Závery experimentálnych prác poukazujú na prítomnosť vysokých koncentrácií oxidačné aktívnych molekúl, ktoré sú medziproduktami oxidačno-redukčných reakcií v poškodených tkanivách v súvislosti s prítomnou neuropatickou bolesťou (37, 38). Oxidačný stres môže vyvolať negatívne zmeny najmä v centrálnom nervovom systéme vzhľadom na vysokú zraniteľnosť neurónov a gliových buniek voči oxidačným vplyvom a metabolickým zmenám, ktoré prebiehajú v rámci neurozápalu. Z experimentálnych výskumov vyplýva, že oxidačný stres hrá dôležitú úlohu v rámci viacerých mechanizmov, ktoré sú zapojené do nociceptívnej modulácie, centrálnej senzitizácie a priebehu neurozápal ovej odpovede. Vo všeobecnosti zápal predstavuje základnú biologickú reakciu, ktorá je prísne regulovaná súhrou rôznych mechanizmov, pričom jej cieľom je obnovenie integrity tkaniva. Neurozápal je forma zápalovej reakcie, ktorá sa týka periférnej a centrálnej nervovej sústavy. Je charakterizovaný zvýšenou vaskulárnou permeabilitou, infiltráciou tkaniva imunitnými bunkami, aktiváciou gliových buniek, šírením genetickej proinflamačnej informácie makrofágmi do iných častí nervového systému a jeho okolia a zvýšenou produkciou zápalových mediátorov v nervovom tkanive. Dôležitým prooxidačným enzýmom v neurozápalových reakciách je NADPH oxidáza (NOX), ktorá katalyzuje premenu kyslíka na superoxidový anión radikál podľa rovnice: NADPH + 202 <-> NADP+ + 20~2 + H+ Ďalším významným enzýmom je enzým NO-syntáza (NOS) katalyzujúca reakciu, ktorej výsledkom je reaktívna forma dusíka (RNS), NO. Tieto molekuly, podobne ako reaktívne 64 formy kyslíka, majú okrem významných prooxidačných vlastností aj dôležitú signalizačnú funkciu. V rámci patofyziologických ciest vzniku neuropatickej bolesti NOS zvyšujú excitabilitu nervových vlákien, a to prevažne nepriamo, cestou mitochondriálneho poškodenia (38, 39). Anti-inflammatory cytokines t p. Antioxidant ► defenses Potential therapeutic strategies —► Promotion I induction —) Inhibition Production Obrázok č.3: Prehľad interakcií a vzájomných vzťahov medzi neurozápalovými mechanizmami a oxidačným stresom pri rozvoji chronickej bolesti. TLR - Toll-like receptor, P2R - Purinérgny 2 receptor, MAPK - mitogénom aktivovaná proteinová kináza, NF-kB -nukleárny faktor kappa B (Zdroj: Teixeira-Santos 2020). Zápalové reakcie sú riadené komplexnou sieťou regulačných mechanizmov, ktoré kontrolujú priebeh, intenzitu a supresiu škodlivých dôsledkov zápalu. Za fyziologických okolností je zápalová kaskáda po dosiahnutí obranných alebo reparačných cieľov potlačená biochemickými a imunitnými mechanizmami. Ak však tieto mechanizmy regulácie zlyhajú, môže vzniknutá situácia viesť k dlhodobej a nekontrolovateľnej zápalovej reakcii, čo má za následok rozvoj chronických stavov, medzi ktoré patrí aj rozvoj chronickej neuropatickej bolesti. Pribúdajúce dôkazy poukazujú na to, že v procesoch regulácie zohrávajú úlohu viaceré významné molekuly akými sú špecializované „pro-resolving" mediatory, medzi ktoré patria deriváty odvodené od kyseliny arachidonovej (lipoxíny) a mediatory odvodené od polynenasýtených mastných kyselín: kyselina eikozapentaénová, kyselina dokozapentaénová a kyselina dokozahexaénová (resolvíny, maresíny a protektíny). Tieto molekuly sú významné inhibitory NOX a ich deficit sa spája s vyššou intenzitou neurozápalovej odpovede (38, 39). 65 Liečba chronickej bolesti je komplexná. Opiera sa o komplexnú farmakologickú, intervenčnú a nefarmakologickú liečbu, a však naďalej sa hľadajú ďalšie efektívne možnosti terapie. Nové experimentálne štúdie poukazujú na možnosti ovplyvnenia neurozápalu aj ďalšími cestami v rôznych fázach zápalovej reakcie. Tieto terapeutické prístupy je možné rozdeliť do dvoch hlavných línií, z ktorých každá sa zameriava na odlišné fázy zápalového procesu. Intervencie konvenčnej protizápalovej liečby sa primárne zameriavajú na inhibíciu prozápalových mediátorov za účelom zmiernenia symptómov a obmedzenia zápalovej reakcie. Jedná sa o aplikáciu potentných antioxidantov v priebehu celej periódy zápalovej reakcie. Stratégie „pro-resolution": Na rozdiel od nešpecifickej všeobecnej liečby, stratégie „pro-resolution" sú zamerané na protizápalové procesy, uľahčenie včasnej obnovy tkanivovej homeostázy a minimalizáciu rizika vzniku chronického zápalu. Do tejto línie liečby sa radí skupina tzv. pro-resolving mediátorov (PRM), ktoré pôsobia ako aktívne inhibitory syntázy oxidu dusnatého (NOX). PRM majú schopnosť supresiou modulovat' zápalovú odpoveď a tým brániť možnej chronifikácii bolesti. Onset phase I Resolution phase I Proinflammatory phase Self-limited protective response Failed resolution > Therapeutic strategies Induction of the resolution phase Inhibition / suppression of inflammatory mediators Time Homeostasis restoration Chronic inflammation Obrázok č.4: Zobrazenie jednotlivých fáz neurozápalovej odpovede v patogenéze neuropatickej bolesti (Zdroj: Teixeira-Santos, 2020). 66 Ďalšou významnou antioxidačnou molekulou, ktorá potláča nežiadúce oxidačno-redukčné deje v rámci neurozápalu a pozitívne ovplyvňuje priebeh chronifikácie bolesti je glutatión. Glutatión hrá kľúčovú úlohu pri ochrane nervových buniek pred poškodením a degeneráciou. Pomáha udržiavať štrukturálnu integritu a funkciu neurónov, čím predchádza alebo zmierňuje poškodenie nervov spôsobené rôznymi noxami, akými sú napríklad oxidačný stres, zápalové reakcie a neurotoxické látky. Zachovaním fyziologickej integrity nervových štruktúr môže glutatión zmierniť symptómy neuropatickej bolesti a podporiť regeneráciu nervov Ďalším mechanizmom, ktorým glutatión nepriamo ovplyvňuje moduláciu a percepciu bolesti je zapojenie sa do regulačných procesov glutamátergného prenosu. Glutamát je kľúčový neurotransmiter zapojený do signalizácie bolesti a dysregulácia glutamátergného prenosu prispieva k rozvoju neuropatickej bolesti. Ukázalo sa, že glutatión moduluje hladiny a aktivitu glutamátu, čím ovplyvňuje vnímanie a prenos bolesti. Reguláciou glutamátovej neurotransmisie môže glutatión zmierniť hypersenzitivitu neuropatickej bolesti. Glutatión sa ďalej podieľa na regulácii endogénnych systémov modulácie bolesti, ako je napríklad endogénny opioidný systém. Zvýšením aktivity týchto systémov môže glutatión prispieť k zmierneniu symptómov neuropatickej bolesti (38, 39). Prehľadový článok o charakteristike glutatiónu a jeho potenciálnych terapeutických účinkoch publikoval náš lekársko-vedecký tím v časopise Molecules Q2, IF 4.6. • Vašková J, Kočan L, Vasko L, Perjési P. Glutathione-Related Enzymes and Proteins: A Review. Molecules. 2023 Feb 2;28(3):1447. doi: 10.3390/molecules28031447. PMID: 36771108; PMCID: PMC9919958. Vplyv oxidačného stresu a možnosti jeho supresie pri rozvoji neurozápalu, neuropatickej bolesti a pooperačnej kognitívnej dysfunkcie u pacientov po kardiochirurgických a nekardiochirurgických výkonoch skúmame v aktuálne prebiehajúcej prospektívnej observačnej klinickej štúdii NeuOX-postSurg Trial NCT06391866. 67 molecules Review Glutathione-Related Enzymes and Proteins: A Review Janka Vašková lf* , Ladislav Kočan 2, Ladislav Vaško 1 and Pál Perjési 3'* 1 Department of Medical and Clinical Biochemistry, Faculty of Medicine, Pavol Jozef Šafárik University in Košice, 040 11 Košice, Slovakia 2 Clinic of Anaesthesiology and Intensive Care Medicine, East Slovak Institute of Cardiovascular Disease, 040 11 Košice, Slovakia 3 Institute of Pharmaceutical Chemistry, University of Pees, 7600 Pecs, Hungary * Correspondence: janka.vaskova@upjs.sk (J-V.); pal.perjesi@gytk.pte.hu (P.P.); Tel.: +42-155-234-3232 (J.V.) Abstract: The tripeptide glutathione is found in all eukaryotic cells, and due to the compartmental-ization of biochemical processes, its synthesis takes place exclusively in the cytosol. At the same time, its functions depend on its transport to/from organelles and interorgan transport, in which the liver plays a central role. Glutathione is determined as a marker of the redox state in many diseases, aging processes, and cell death resulting from its properties and reactivity. It also uses other enzymes and proteins, which enables it to engage and regulate various cell functions. This paper approximates the role of these systems in redox and detoxification reactions such as conjugation reactions of glutathione-S-transferases, glyoxylases, reduction of peroxides through thiol peroxidases (glutathione peroxidases, peroxiredoxins) and thiol-disulfide exchange reactions catalyzed by glutaredoxins. Keywords: cell; redox homeostasis; glutathione; glutathionylation; glutathione system; glutathione enzyme check for updates Citation: Vnskova, J.; Kocan, L, Vasko, L.; Perjesi, P. Glutathione-Related Enzymes and Proteins: A Review. Molecules 2023, 28,1447. https://doi.org/10.3390/ molecules28031447 Academic Editor: Hyun-Ock Pae Received: 15 January 2023 Revised: 30 January 2023 Accepted: 31 January 2023 Published: 2 February 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1. Introduction Glutathione (GSH) was first isolated in 1888 by De-Rey-Pailhade. He named the substance phylothion, the Greek expression for sulfur loving [1], Its structure was controversial for several years. Initially, it was described as a sulfur-containing dipeptide [2]. Later the structure was refined, demonstrating that the substance is a tripeptide, y-Glu-Cys-Gly [3-5]. Other related compounds, such as y-Glu-Cys-Gly-spermidine and (y-Glu-Cys)„-Gly in £. coli and plants, were also described [6]. The thiol group of the cysteine residue enables GSH to function as both a reducing agent and a nucleophilic center [7]. Glutathione occurs in two free forms: the reduced (GSH) thiol and the oxidized (GSSG) disulfide forms (Figure 1). In addition, it can be bound to proteins and other thiols, affecting their activity. In its reduced and oxidized forms (GSH, GSSG), glutathione is ubiquitous in mammalian cells ranging in 1-10 mM concentrations [8]. Under physiological conditions, more than 98% of total GSH occurs in the reduced form [9,10]. It is an essential antioxidant against reactive oxygen and nitrogen species [11]. The compound plays a critical role in maintaining the redox homeostasis of the cells and in cell cycle regulation, apoptosis, immunological defense, and pathological abnormalities [8]. Furthermore, it is one of the endogenous substances involved in the metabolism of endogenous (e.g., estrogens, leukotrienes, prostaglandins) and exogenous compounds (e.g., drugs, non-energy-producing xenobiotics) [12]. These latter transformations could be the molecular basis for eliminating foreign substances from the body. In this review, the role of glutathione and glutathione-dependent enzymes in the maintenance of redox homeostasis is summarized. Molecules 2023, 28, 1447. https://doi.org/10.3390/molecules28031447 https://www.mdpi.com/journal/molecules Molecules 2023, 28, 1447 2 of 22 Figure 1. Structure of reduced (GSH) and oxidized (GSSG) forms of glutathione. 2. Glutathione Together with glutaredoxins (Grx), GSH acts to reduce disulfide bonds and is, in turn, oxidized to glutathione disulfide (GSSG), which is reduced by NADPH-dependent glutathione reductase. The GSH/GSSG, NADPH/NADP+, Grx-SH/Grx-SS, and Trx-SH/Trx-SS are the most important redox couples in maintaining cellular redox homeostasis [13]. The standard apparent redox potential (E'°) of GSH is -288 mV (pH 7, 298.15 K, 0.25 M ionic strength), which is well between the most negative HVH2 (—423 mV) and the most positive, O2/H2O (+849 mV) redox couples [14]. Accordingly, the GSH/GSSG redox couple can readily interact with most physiologically relevant redox couples, undergoing reversible oxidation or reduction [7], Given the availability of glutathione in the cells, the reactions of protein thiols are mediated by multiple enzymes and enzyme systems, thus allowing it to participate in the abovementioned functions and regulatory pathways. Among them are glutaredoxins, which are central in the response against oxidative stress as the biological activity of many proteins are modified by the formation of GSH-mixed disulfides. Furthermore, other redox-maintaining enzymes such as glutathione peroxidases, and detoxification enzymes, glyoxylases, are closely related to carbohydrate metabolism [15,16]. Thus, the involvement of glutathione and its activity in the cell represents a wide range of biological and biochemical processes. The consequence of its deficiency results in increased stress conditions, which is the basis of the pathophysiology of many organ or tissue-specific diseases such as inflammation, virus infections (HIV), sickle cell anemia, cancer, diabetes, heart attack, stroke, liver disease, cystic fibrosis, Alzheimer's, and Parkinson's disease [17,18]. 2.1. The Role of the Liver in Glutathione Synthesis and Distribution Synthesis of GSH occurs in the cytoplasm in all cells in two subsequent ATP-dependent reactions catalyzed by glutamate-cysteine ligase and GSH synthetase, from where it is transported to other organelles and extracellular space [8,19]. Glycine, glutamate, and cysteine as nonessential amino acids can be obtained from dietary sources or synthesis. The liver removes a significant amount of resorbed cysteine from the portal vein [20], However, cysteine can be synthesized by methionine transsulfuration in the liver [21]. The liver is responsible for the metabolism of up to half of the daily methionine intake, predisposing the liver to almost exclusive transsulfuration activity and being the most important in interorgan GSH homeostasis [22]. Thus, a considerable amount of GSH is produced by the liver and released into plasma and bile [22], Rat liver cytosolic GSH has a half-life of 2-3 h [8], and the daily turnover for GSH is estimated to be higher than cysteine turnover in the body protein pool, around 40 mmol per day [21]. Transsulfuration is not present in the fetus, newborn infants, or patients with cirrhosis [23]. Cirrhosis causes a decrease in methionine adenosyltransferase activity following a reduction in S-adenosylmethionine production and lower effectivity of the transsulfuration pathway [24]. Glutathione concentration within extracellular fluids and blood plasma reaches only several uM; however, in some extracellular fluids, such as lung lining fluid, 100-400 \xM levels have been detected [25,26]. Molecules 2023, 2», 1447 3 of 22 2.2. Cell Uptake and Metabolism of Glutathione To date, two mechanisms of glutathione uptake into mammalian cells are known [19]. The most common one is primarily associated with the activity of y-glutamyl transpeptidase (GGT) (Figure 2). GGT is localized to the cell surface and cleaves only extracellular substrates, GSH, and oxidized GSH (GSSG), its most abundant ones. The amide bond between the glutamine y-carboxyl and the cysteine amino units does not allow cleavage of GSH by cellular and circulating serum peptidases [27]. It is hydrolyzed by the y-glutamyltranspeptidase (GGT) to glutamate and Cys-Gly. Cys-Gly can be cleaved by membrane-bound dipeptidases (MDBs) or intracellular Cys-Gly peptidases. Cellular uptake of Cys-Gly or the individual Cys, Gly, and glutamate units serve as precursors for intracellular GSH synthesis. GGT is expressed on the luminal surface of excretive and absorptive cells that line glands and ducts throughout the body, with the highest level of GGT activity in the kidney and pancreas ducts [28]. It is nearly absent, however, from the hepatocytes and cardiac myocytes [7]. The absence of GGT activity on the apical surface of the kidney's proximal tubules by genetic disorder results in glutathionuria [29,30]. extracellular space Figure 2. Involvement of y-glutamyl transpeptidase (GGT), glutathione-S-transferases (GST), their subfamily of Membrane Associated Proteins in Eicosanoid and Glutathione metabolism (MAPEG), and glyoxylases (Glo) in the intracellular metabolism of GSH. MRP1 (multidrug resistance-associated protein 1) transporter facilitates the unidirectional transport of conjugates. GGT has multiple functions, including catalytic transfer of y-glutamyl groups to amino acids and short peptides, hydrolysis of GSH to glutamyl moiety and cysteinyl glycine, and catabolism of GSH conjugates [31]. GGT allows hydrolysis of a broad range of y-glutamyl amides and transpeptidation of amino acids or dipeptides [32], So GSH, its S-conjugates, GSSG, y-glutamyl di- or tripeptides, glutamine, 1-a-methyl derivatives of y-glutamyl amides, various lipid-derived mediators (e.g., leukotriene C4), geranylgeranyl, poly-y-glutamyl derivatives serve as substrates of GGT [33-36]. Many tumor cells express GGT on their entire cell surface and can therefore cleave GSH not only in the ductal but also in interstitial fluid and blood [37]. GGT expression provides tumor cells with an additional source of cysteine and cystine from the breakdown of extracellular GSH and GSSG [38]. Besides the GGT pathway, there is evidence of Na+-dependent and Na+-independent glutathione transport systems for glutathione cell uptake expressed in the renal basolateral membrane [38,39], the small intestine [40], and the brain [41 ]. In the renal basolateral membrane, two Na+-independent Organic Anion Transport systems (OAT1 and OAT3) [42] and the Na+-dependent dicarboxylate carriers are the most important organizations [43,44]. On the other hand, the plasma membrane glutathione efflux can be facilitated by specifically or ubiquitously expressed membrane proteins and anion channels such as multidrug resistance-associated proteins (MRP1-5), Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), Arginine/Ornithine Transport ATP-binding Proteins (OATP 1,2), and ATP-Binding Cassette superfamily G member 2 (ABCG2) [19]. Molecules 2023, 2», 1447 4 of 22 2.3. Intracellular Distribution and Functions ofGSH Within the cell, there are three main glutathione pools. The cytosol (80-85%), the mitochondria (10-15%), and the endoplasmic reticulum [45-47], Studies by Birk et al. and Montero et al. [48,49] pointed out that the total glutathione content in the lumen of the endoplasmic reticulum even exceeds the entire cellular glutathione content. GSH and GSSG concentrations depend on the subcellular compartment, the cell type, and the organism. Accordingly, the redox potential of the GSSG/2GSH system varies from tissue to tissue, from organism to organism. This relies on the proportion of GSH and GSSG and the total concentration of glutathione, which is quite challenging to estimate their actual concentration and ratio in vivo [50,51], For example, taking the local pH and GSSG/2GSH ratios into consideration, cytosolic EpH7.o = —289 mV (or even lower), mitochondrial matrix EpH7.o = —296 mV (or even lower), and human plasma EpH7.4 = —118 mV half-cell reduction potentials (£(,c) have been estimated [52]. Furthermore, a correlation has been found between the cell cycle, the condition of the cell (stressed, apoptotic, etc.), and the GSSG/2GSH ratio. For instance, in cell proliferation (£|lc = —240 mV), in cell differentiation (Ehc = —200 mV), and in apoptosis (Ehc = —170 mV), which can be applicable for a better understanding of oxidative stress [13,53]. Van't Erve et al. [54] found that GSSG/2GSH levels and reduction potential in erythrocytes reflect genetic differences between individuals. Cytoplasmic glutathione levels impact glutathione diffusion through nuclear pore complexes [55], playing a role in oxidative signaling during proliferation, epigenetic control of histone activity, and the cell cycle control, mainly in the S + G2/M phase [56,57]. ATP-dependent transporters have also been reported to import glutathione into the nucleus [58], Glutathione synthesis occurs only in the cytosol; thus, the mitochondrial pool is supplied by GSH transport and maintained by reducing its oxidized form via the activity of glutathione reductase. Glutathione passes the mitochondrial outer membrane through the mitochondrial porin, a voltage-dependent anion channel (VDAC). As a negatively charged molecule, glutathione cannot diffuse through the mitochondrial inner membrane. Its transport into the mitochondrial matrix is either active or provided in exchange for another anion [7]. Six of the eight anion carriers have the potential for GSH import through the inner membrane into mitochondria. Monocarboxylate, dicarboxylate (DIC), 2-oxoglutarate (OGC), tricarboxylate (or citrate), glutamate-hydroxide, glutamate-aspartate transporters involved in the transport of GSH also provide intermediates of the Krebs cycle and the gluconeogenesis pathway [59]. DIC and OGC were identified as major GSH transporters, although at the expense of Krebs cycle intermediates [60]. Around 70-80% of GSH transport could be associated with DIC and OGC activity in the kidney, but only about 45-50% of liver mitochondria [61]. DIC imparts malate (malonate or succinate) in exchange for phosphate, sulfate, and thiosulfate. Malate conversion into oxalacetate, followed by the formation of phosphoenolpyruvate, is limited for gluconeogenesis in the cytosol. Reduction in DIC expression leads to decreased glutathione levels and impaired complex I activity [62]. OGC transfers 2-oxoglutarate substituting dicarboxylate [63], thus regulating respiration and glycolysis. While succinate from the matrix side increases the affinity of OGC to malate, substrates such as phenyl succinate, pyridoxal phosphate, retinoic acid, and ethanol cause inhibition of OCG. Reduced activity of OCG leads to lower energy production, increased oxidative stress, and it could be the basis of liver or nervous tissue diseases [64-66]. GSSG is not transported out from mitochondria [67]. The endoplasmic reticulum offers a unique setting concerning GSH homeostasis. It contains the thiol oxidase Erol, which catalyzes the formation of disulfides transmitted to folding substrates via protein disulfide isomerase (Pdil). Both reduced and oxidized forms of glutathione are transported into the endoplasmic reticulum at different rates, with a preference for the reduced form [68]. Since GSH is oxidized but not reduced in the ER, GSH must be imported into the ER, while GSSG is exported to the cytosol [69]. A study by Ponsero et al. [70] brought up the finding of facilitated diffusion of GSH through the Sec61 protein translocation complex. In the sarcoplasmic reticulum, ryanodine receptor Molecules 2023, 2», 1447 5 of 22 calcium channel type 1 (RyRl) was suggested to play an important role [71]. However, Bachhawat et al. [19] pointed out that this might result from the S-glutathionylation of several cysteine residues within the RyRl molecule. To maintain GSH homeostasis, part of GSSG is transported to the cytosol through vesicular transport [72]. Most GSSG reacts with proteins or protein disulfide isomerase involved in oxidative protein folding [64]. A lower GSH:GSSG ratio results in more oxidizing conditions (—240 mV) [70] in the endoplasmic reticulum allowing protein disulfide formation. GSH plays an essential role not only in the peripheral tissues but in the central nervous system (CNS) as well. Brain tissues are rich in unsaturated fatty acids. Due to their relatively low levels of antioxidants or antioxidant enzymes, they are rather sensitive to oxidative damage. The most important small molecular CNS antioxidants are GSH, ascorbic acid (vitamin C), and tx-tocopherol (vitamin E) [73]. Among these antioxidants, GSH seems to be the determining agent because it is selectively decreased in the brains of patients with these neurodegenerative diseases (e.g., Parkinson's disease, Alzheimer's disease, and Amyotrophic lateral sclerosis) [74]. Therefore, regulating the redox state by intracellular GSH is crucial for maintaining cellular functions under physiological and pathological conditions. In the central nervous system, besides the functional neurons, there are several other types of cells for the nervous system to function properly. This is where a set of glial cells intervene, which make up 25-50% of the nerve mass [75]. The most common type of glial cells in the CNS are the astrocytes and the microglia. Synthesis of GSH occurs both in the neurons and the glial cells. In an early work by Rice and Russo-Menna (1998) [76], GSH levels of glutathione in neurons and glia were reported to be 2.5 nM and 3.8 mM, respectively. The authors found that ascorbate predominates in neurons (10 mM), whereas GSH is slightly predominant in glia. According to the above, GSH supplementation seems promising for treating patients with neurodegenerative diseases. 2.4. Acid-Base Properties The acid-base properties of glutathione (GSH) have long been the focus of scientific interest. It has three acidic (thiol, glycinyl carboxyl, glutamyl carboxyl) and one basic (amino) functional group. Accordingly, in an aqueous solution, glutathione can exist in four different macroscopic protonation states: L3" & HL2- & H2L" h H3L & H4I + where L3~ is the fully deprotonated, H4L+ is the fully protonated GSH molecule. Since the HL2- and the H3L forms have four protonation isomers (microspecies) each, and the H2L~ form has six microspecies, the molecule has sixteen different protonation states (microspecies) altogether [77]. The micro and sub-micro protonation constants characterize the acid-base properties at the submolecular level [78]. These constants allow quantification of the proton binding capacity of submolecular basic units when the protonation states of all other sites are defined in the molecule [79]. Group constants are special micro constants when the rest of the groups in the molecule are far enough apart, and their protonation does not affect the basicity of the group [80]. The rotational state of the flexible parts of the molecules is defined by the sub-micro constants when protonation occurs [81 ]. The correct characterization of the basicity of the sites of protonation of multidentate ligands can be conducted using the micro and sub-micro constants. In addition, this group of constants is used to measure the concentration of different protonation forms, of which the principal form is not always the reactive form in chemical and biological processes. [82-86]. The macroscopic protonation constants (K1-K4) determined by 1H NMR-pH titrations were as follows: logKl 9.65; logK2 8.78; logK3 3.52; and logK4 2.22 [77]. The obtained values were found to be very similar to those determined in earlier works of Pirie and Pinhey [87] (9.62, 8.66, 3.53, 2.12), Li et al. [88] (9.65, 8.75, 3.59), and Molecules 2023, 2», 1447 6 of 22 Martin and Edsall [89] (9.62, 8.74). The results demonstrated that the first and the second protonation constants were predominated by the overlapping protonation of the amino and the thiolate site, the amino being typically more favored. The carboxylate groups also protonated in an overlapping fashion, the glycinyl carboxylate being more basic. It is worth mentioning that the protonated amino group makes the inherently more basic glutamyl carboxylate more acidic [77]. It is important to note that the physico-chemical properties (e.g., complex formation, nucleophilic reactivity, redox properties) and biological functions of glutathione could be significantly different at different protonation states (i.e., in solutions with different pH values) [90-93] and its redox behavior [94,95]. Furthermore, ionic strength and the nature of ionic media also affect the acid-base characteristics of glutathione [96], 2.5. Antioxidant Properties The pKa value of GSH (ranging from 8.6 to 8.8 [87-89] results in low thiol reactivity in the cellular environment [97]. Still, high GSH concentrations enable some reducing activities against oxidizing agents in the cell [98]. GSH, for example, can reduce H2O2, resulting in GSSG and water [99], The rate of reaction depends on the cellular GSH level and the ratio of GSH to H2O2 concentrations [100]. Recently, Zinarullina et al. [101] confirmed that the oxidation of GSH is accompanied by radical formation. GSH reacts with the majority of free radicals generating thiyl radicals. Consecutive reactions of the radicals with a thiolate anion and molecular oxygen lead to disulfide and superoxide radicals formation [102]. Furthermore, y-glutamylcysteine, a GSH precursor, was found to decompose H2O2 similarly to glutathione peroxidase-1 [103]. Glutathione exists in 100 uM concentrations as glutathione persulfide (GSSH) [104], the latter exhibiting higher activities due to its higher nucleophilic power than GSH [105], Under specific conditions, GSSH reacts with H2O2, while GSH does not [106]. Furthermore, its reactions with one-electron oxidants are faster than similar reactions of thiols [107]. GSSH are intermediates in the synthesis of iron-sulfur clusters and mitochondrial H2S oxidation [108-110]. GSH can react with HS~ catalyzed by sulfide quinone oxidoreductase or thiosulfate sulfurtransferase, forming GSSH, which can reduce oxidized thioredoxin. Single-domain sulfurtransferase (TSTD1, known as rhodanese) and mercapto pyruvate sulfurtransferase can also directly transfer sulfides to GSH and the thioredoxin antioxidant systems [111]. Mutations in persulfite dioxygenase, oxidizing GSSH to sulfite and GSH, are bases for autosomal-recessive inherited ethylmalonic encephalopathy [112]. 2.6. Redox Signaling Properties Signaling is the process that makes cells capable of reacting to the change in their environment (intercellular signaling) or their homeostasis (intracellular). The initial step of the process is the interaction of the signaling particles (ligands) with the target molecule (receptor). The well-known signaling mechanisms involve protein-protein interactions, allosteric changes induced by the binding of ligands, proteolytic processing, and chemical modifications such as acylation, acetylation, alkylation, and phosphorylation of proteins. On the contrary, redox signaling is the transduction of signals based on the transfer of electrons. Redox signaling involves a broad spectrum of pathways involving free radicals, redox-active metals (e.g., iron, copper), or reductive equivalents [74]. Here only those pathways are mentioned that are based on a modification of signaling proteins through the modification of one amino acid, cysteine. The physiological level of hydrogen peroxide) (H2O2) and nitric oxide (NO) can selectively react with the thiol function of the cysteinyl residues at the active site of the proteins (receptors, enzymes, transporters, etc.). Accordingly, the receptor-mediated stimulation of the H2O2 and NO production are part of normal physiology; this is especially true for the longer-lived H2O2. [113]. However, overproduction of these and related species (ROS and RNS) lead to irreversible oxidation of the thiol residues and impairs cellular protein functions [114,115]. The GSSG/2GSH redox system is fundamental in the cells and, together Molecules 2023, 2», 1447 7 of 22 with other redox-active couples (including NADPH/NADP+, Trx-SH/Trx-SS), regulates and maintains the appropriate cellular redox status. For example, the GSSG/2GSH half-cell reduction potential differed in cell proliferation, differentiation, and apoptosis [13,53]. Thus, changes in the GSSG/2GSH ratio are fundamental in controlling signal transduction that supports cell cycle regulation and other cellular processes [55]. The functions and activities of GSH as the main regulator of cellular redox status and redox signal transduction have been reviewed [17,116-119]. GSH acts protectively against oxidative stress by reacting directly with NO, superoxide anion radical (CV-), H2O2, hydroxyl radical (OH), peroxinitrite anion (ONOO~), and the lipid peroxidation product 4-hydroxy-2-nonenal (4-HNE) [116,117]. Such reactions directly modify the cellular GSSG/2GSH half-cell potential, a physiological signaling event. Furthermore, changing the GSH level results in a selective change in the activity of the thioredoxin/glutathione systems [118], the glutaredoxin/glutathione system [119], and the activity of some GST isoforms. The latter protein family is involved not only in the metabolism of xenobi-otics but also of endogenous compounds which play critical roles in regulating signaling pathways [120-122]. 2.7. Reactions with Electrophilic Xenobiotics Glutathione-S-transferases (GST) lower the pKa of GSH thiol under 6, enhancing rates of nucleophilic addition and substitution reactions with electrophylic xenobiotics (Figure 2). These reactions are examples of Phase II bioconjugation reactions, most of which result in reduced toxic effects of the parent compounds or their metabolites [98,123]. Other enzymes/enzyme systems, e.g., selenium-containing glutathione peroxidases (GPx) or per-oxiredoxins (Prdx), use GSH to reduce various peroxides and hydroperoxides. Glyoxalase (Glo) performs conjugation of GSH with the glycolysis byproduct methylglyoxal to form (S)-lactoylglutathione (Figure 2). Moreover, glyoxalase II (Glo-2) catalyzes S-glutathionylation using (S)-lactoylglutathione [124]. 3. The Glutathione Peroxidase System The glutathione or glutathione peroxidase system consists of glutathione peroxidase (GPx) and glutathione reductase (GR). In the decomposition reaction of H2O2 or other organic peroxides (HOOR), two molecules of GSH reduce the substrate to H2O or the corresponding alcohol (HOR) and restore the enzyme forming GSSG with concomitant formation of GSSG and H2O. 2 GSH + HOOR -» GSSG + HOR + H20 (1) GSSG can be excreted from the cell or recycled by GR using the reducing power of NADPH (Figure 3). NADPH arises in two reactions of the pentose phosphate pathway, which is the most potent source of it. However, NADPH can also be formed directly in the mitochondria by NAD(P)+ transhydrogenase, mitochondrial/cytosolic NADP-dependent isocitrate dehydrogenase, or cytosolic malate dehydrogenase [125]. GR is a homodimeric flavoprotein consisting of 52 kD monomers. Except for synthesis, the activity of GR represents a second source of GSH in the cytosol and some organelles, such as mitochondria. Although inhibition of GR has been reported to cause a depletion of GSH and accumulation of GSSG [126], a comprehensive study of the GR and the cellular thiol redox system is missing [127]. Inhibition of the enzyme has also been related to the toxicity of various chemicals and metals [128,129]. Molecules 2023, 2», 1447 8 of 22 . Pr-SH GSSG NADPH ♦ H' GSSG » H30 + OJ LOH ♦ HjO ^S NADPH * H- Figure 3. Basic reaction mechanisms of glutathione peroxidase (GPx), and glutaredoxin in (de)glutathionylation using (GSH)GSSG, respectively, and reduction of GSSG by the activity of glutathione reductase (GR) with reducing the power of NADPH + H+. Reduction of peroxiredoxin (Prdx) after disposal of peroxides is ensured by thioredoxin (Trx), which is reduced by consumption of NADPH + H+ in catalytic efficiency of thioredoxin reductase (TrxR). The term glutathione peroxidase (GPx) describes only a small subgroup of the peroxidases [130], which belong to a group of phylogenetically related enzymes. GPx 1—4 are selenoproteins with selenocysteine (SeCys) in the catalytic center. GPx6 is a human selenoprotein [131]. Their important antioxidant function was shown in various places and cell structures: GPxl is ubiquitous in the cytosol and mitochondria, GPx2 in the intestinal epithelium, and GPx3 in the plasma; all three work in the aqueous phase reducing H2O2 and free fatty acid peroxides [131]. GPx4 protects mainly membranes by reducing phospholipid and cholesterol peroxides [131,132]. Gpx5, which contains cysteine instead of Se in the active center, is a secretory enzyme of the epididymis. GPx6 is a human selenoprotein and is formed by the olfactory epithelium. GPx7 and GPx8 are also CysGPx with low peroxidase activity. GPxl, 2, 3,4,5, and 6 are homotetramers, which could determine their specificity for hydrogen peroxide. GPx4, 7, and 8 are monomers. This structure probably enables the reaction with more complex lipid hydroperoxides, but this has been proven only for GPx4 [132]. The catalytic center of GPx was first characterized as a triad consisting of SeCys or Cys, Gin, and Trp. It was later found to be a tetrad with Asn. A conservative feature for these GPx is the presence of a second or even a third cysteine residue. The reaction mechanism differs between individual GPx isoforms, whose activity requires GSH. In general, they do not form a ternary complex between the enzyme, hydroperoxide, and GSH, but the reaction has a concomitant oxidation and a reduction part. In the oxidation part, deprotonation takes place in the same way. The side chains of the Glu, Try, and Asp residues form a highly nucleophilic region in the enzyme's active center, where oxidation of the active site selenocysteine (RSeH) or cysteine (RSH) occurs after binding the peroxide. This reaction results in the formation of a selenenic acid (RSeOH) derivative. The selenenic acid is then converted back to the selenol (RseH) by a two-step process that begins with a reaction with GSH to form the GS-SeR and water. A second GSH molecule reduces the GS-SeR intermediate back to the selenol, releasing GS-SG as the byproduct [52,132]. A simplified representation (with H2O2 as a substrate) is shown below: RSeH + H202 -» RSeOH + H20 (2) RSeOH + GSH -> GS-SeR + H20 (3) GS-SeR + GSH -» GS-SG + RSeH (4) Glutathione reductase then reduces the oxidized glutathione to complete the cycle: GS-SG + NADPH + H+ -> 2 GSH + NADP+ (5) Molecules 2023, 2», 1447 9 of 22 Selenium deficiency results in increased GSH synthesis in the liver with accompanying release to the plasma [133]. Increased plasma GSH led to cysteine depletion, impaired protein synthesis, decreased GPx, and increased GST activities [134]. Usually, GPx requires GSH in millimolar concentrations in the intracellular space, and plasma GSH reaches micromolar concentrations, which questions GPx's antioxidant function [135]. However, within the cell, in the cytosol and mitochondria, the GPx system appears to be very efficient in the elimination of H2O2 due to the low (100-200 pM) Km value of the enzyme [136] and the range of substrates [137]. Mimicking GSH, y-glutamylcysteine can be used by GPxl as a cofactor [103]. 4. Glutaredoxins (Grx) The thiol oxidoreductase glutaredoxins (Grx) are small proteins reducing various protein disulfides (PrSSPr) and GSH-protein mixed disulfides (PrSSG), where the electron donor is glutathione [138]. Grxs catalyze glutathionylation, post-transcriptional modifications, and the disulfide exchange between GSSG and protein thiols (PrSH) [139] (Figure 3). Grx-catalyzed (de)glutathionylation is an important event in signal transductions and serves as the primary protective mechanism against the irreversible oxidation of cysteine residues [115]. As mentioned above, the standard cell potential changes depending on the environment and the cell itself. Cell proliferation occurs at approximately —240 mV, differentiation at about —200 mV, and apoptosis at around —170 mV [55]. Changes in the GSH/GSSG redox potential can be sensed by Grxs, which operate as GSH-dependent reductases at about —240 mV and GSSG-dependent oxidases at about —170 mV [140]. Grxs are characterized by their active site motif. Dithiol-type Grx (class I) enzymes have a Cys-Pro-Tyr-Cys active site, while monothiol Grx (class II) enzymes do not contain a thiol at the C-terminus of the active site (Cys-Gly-Phe-Ser). Dithiol Grxs and monothiol Grxs with one Grx domain are found in all living organisms. Multi-domain monothiol Grxs (PICOTs, PKC-interacting cousin of thioredoxin) are present in eukaryotic cells. These contain an N-terminal Trx-like domain and three C-terminal monothiol Grxs domains [141], Two other regions were recognized near the active site, the Grx characteristic motif GG and the TVP, which are involved in binding GSH [142], 4.1. Glutathionylation Glutathionylation involves the reversible attachment of glutathione to cysteine residues in target proteins. Conditions of elevated oxidative stress increase the levels of protein glutathionylation. The glutathionylation/deglutathionylation cycle is viewed as a process that acts primarily against ROS/RNS via reducing aberrant cysteine modifications and thereby preventing the formation of damaging irreversible cysteine modifications. There are three pathways of glutathionylation. (a) The thiol-disulfide exchange between GSSG and PrSH is accomplished at a low GSHGSSH ratio. The reactivity of PrSH depends on the thiol pKa [143]. (b) The oxidation of the PrSH yields a thiyl radical (RS), which reacts with the deprotonated form of glutathione (GS~), forming a mixed disulfide radical (RSSG ). After the loss of an electron, a mixed disulfide (RSSG) and a superoxide anion radical (O2'-) are formed [144]. (c) Mixed disulfides can also be formed with low molecular weight thiols with indistinct biological relevance. As Lushchak [60] discussed, inhibition of glutathione reductase, phosphofructokinase, fatty acid synthase, or activation of fructose-l,6-bisphosphatase by CoASSG was shown. Cysteine residues of proteins with a low pKa are targets for redox modulation under oxidative or nitrosative stress conditions. The primary products of these oxidative transformations are the respective thiyl radicals (PrS). These reactive intermediates can react with glutathione (GSH) to form stable glutathionylated protein disulfides (PrSSG) to prevent their further oxidation with molecular oxygen. The protected protein thiol can be regenerated by the deglutathionylation process (e.g., through a reaction with another GSH molecule). Under oxidative stress, the thiyl radical can be further oxidized to form sulfenic (RSOH), sulfinic (RSO2H), or sulfonic acid (RSO3H) derivatives of the Molecules 2023, 2», 1447 10 of 22 proteins. Both sulfenic and sulfinic acids of proteins can be reduced by Trx and sulfiredoxin, respectively [145-147]. In contrast, sulfonic acid cannot be reduced. Both sulfenic and sulfinic acids of proteins can be conjugated to GSH to form S-glutathionylated proteins via glutathione S-transferases (GSTs), Grx, or nonenzymatically. Glutathionylation was referenced to cytoskeletal proteins, metabolic, redox enzymes, cyclophilin, stress proteins, nucleophosmin, transgelin, galectin, and fatty acid binding protein [148], affecting their activity either in activation or decrease. 4.2. Deglutathionylation Deglutathionylation undergoes cleavage of the disulfide linkage of the glutathiony-lated protein with another GSH molecule (Figure 3). The reaction can proceed (a) either in a mixed disulfide intermediate with an N-terminal thiol active site; (b) in a mixed disulfide intermediate by the attack of a second GSH molecule; or (c) by non-covalent binding of the thiol function of both an N-terminal thiol active site and GSH-coordinating metal cofactor in the [Fe-S] binding Grx subgroup [142]. The motif in the active site and the type of disulfide bond in the target protein are decisive for the reaction mechanism [149]. In the reaction mechanism of monothiol Grxs, the reduction of glutathionylated proteins (PrSSG) begins with a nucleophilic attack of the N-terminal cysteine. As a result, glutathionylated Grx and reduced substrate protein are released. The Grx-SG intermediate is cleaved by a GSH molecule, resulting in reduced Grx and GSSG, which is subsequently reduced by GR [150] (Figure 3). In the mechanism of dithiol Grxs, the reduction of PSSG and mixed disulfides begins with a nucleophilic attack of the N-terminal cysteine, but GSH is released. The Grx-protein intermediate is reduced by the second C-terminal active cysteine of Grx, forming oxidized Grx and reduced protein [151,152]. Dithiol Grx can also use monothiol mechanisms. However, both mechanisms are critically dependent on the availability of reduced GSH [153]. Apart from oxidoreductase activity, both classes of Grx proteins can bind [Fe-S] clusters. Class II enzymes are essential in the processes of regulation of Fe metabolism. Their function depends on the [Fe-S] binding capacity and not on the reductase activity [154]. In addition, Grxs have dehydroascorbate reductase and transhydrogenase activity, catalyzing denitrosylation and partial cystine conversion [155]. Monothiol Grxs (Grx3 and Grx5) form an iron-sulfur complex. Both isoforms can transfer iron to specific proteins. However, monothiol Grxs cannot deglutathionylate target proteins [156]. Grx3, localized in the cytosol, has a unique domain structure consisting of an N-terminal Trx-homology domain [141,157]. The first discovered function of Grx3 was related to that of protein kinase C theta, and in T-cells, Grx3 colocalizes with it, hence the name PICOT [157]. Since Grx3 is expressed in a wide variety of organs and tissues, it has been proposed as a redox sensor in signal transduction in response to reactive oxygen and nitrogen species [158]. Nuclear Grx3 has a role in the epigenetic regulation of chromatin by regulating the methylation of myelin transcription factor 1 and cell proliferation [159,160], Grx5 participates in the biogenesis of [4Fe—IS] clusters by interacting with ISCA1 of the mitochondrial homolog of the iron-sulfur cluster assembly and ISCA2 of the cytosolic iron cluster [161,162]. Grx5 forms a cluster in the cytosol with a family of BolA-like proteins (regulatory DNA-binding proteins) for the maturation of iron-sulfur proteins [163]. Grxl and Grx2 are dithiol Grxs. Most human Grxl is found in the cytosol, less in the nucleus [164] and the mitochondrial intermembrane space [165]. Grxl, unlike Trx, is not an essential protein [98]. Grxl activity depends on the redox state of the cells, especially the GSH/GSSG ratio [166]. In addition to deglutathionylation activity, Grxl has also been able to denitrosylate protein Cys-NOs and prevent the pro-apoptotic effect of nitric oxide in tumor cell lines and cardiomyocytes [167,168]. Grx2 is about 20 times less abundant than Grxl [169]. Depending on gene splicing, it is localized in mitochondria, cytosol, or nucleus [170]. Like Grxl, it catalyzes the reduction of disulfides mixed with GSH with a higher affinity but with a lower turnover rate [171]. However, these two proteins behave differently in response to an oxidative environment. While Grxl is inhibited Molecules 2023, 2», 1447 11 of 22 when other structural cysteine residues are oxidatively modified [154], Grx2 is activated. The different response to oxidative conditions is due to the ability of Grx2 to form [Fe-S] clusters [172]. The [Fe-S] clusters act as sensors for Grx2 activity under oxidative conditions [154]. Outside the active site, two cysteines form a [2Fe-2S]-bridged dimer that is enzymatically inactive. Oxidative stress increases GSSG concentration and reduces the availability of GSH for coordination of the [Fe-S] complex, leading to cluster degradation and formation of enzymatically active Grx2 monomers [154]. Grx2 can cycle and accept electrons from thioredoxin reductasel (TrxRl) [171]. In mitochondria, Grx2 has been shown to efficiently catalyze (de)glutathionylation of complex I and SOD1 [173,174]. 5. Peroxiredoxins (Prdx) Peroxiredoxins (Prdxs) are cysteine-dependent peroxidase enzymes [132,175], whose low Km for H2O2 (10 uM) and their ubiquity, comprising up to 0.8% of total protein in some animal cells predispose them for reduction H2O2 [176], However, they can also reduce peroxynitrite, peroxynitrous acid, and lipid peroxides [177,178], Their peroxidatic functions overlap with GPx and catalase, and their catalytic efficiency is lower (~105 M_1 s_1) compared to GPx (~108 M_1 s_1) and catalase (~106 M_1 s_1) [179], Furthermore, comparing Prdx Km for H2O2 with that of GPx and catalase exceeding even the millimolar range [180] suggests that the role of Prdx is rather as a sensor of H2O2 [178] than oxidative stress condition reversal. Prdxs are divided into the subgroups Prdxl/AhpC, Prdx5, Prdx6, Tpx (thiol peroxidase), PrdxQ/BCP, and AhpE. Human Prdxs can be posttranscriptionally modified by glutathionylation, acetylation, ubiquitination, oxidation (RSOH, RSSR, RSO2, RSO3), S-nitrosylation, phosphorylation [181] or tyrosine nitration [182]. Prdxs proceed the same catalytic cycle, where the active site cysteine (peroxidatic cysteine, Cysp) reduces peroxides and forms Cysp-sulfenic acid (RSOH), releasing water or the corresponding alcohol. Some Prdxs contain a second, so-called resolving cysteine (CysR), which reacts with RSOH forming disulfide (Cysp-S-S-CyspJ and water [183]. CysR can originate from the adjacent monomer, the same monomer, glutathione, or a redox-relay binding partner [184]. Accordingly, six human Prdxs isoforms are diversified into three subgroups. In general, the Prdxl subfamily enzymes are the most highly expressed, making up 0.1-1% of the soluble protein in the cell. The "typical 2-Cys" Prdxs are homodimers with two active sites (having both a Cysp and CysR). The disulfide bond is formed between the two subunits in the reaction of RSOH and CysR of the other subunit. Reduction of disulfide bond is catalyzed by Trx (Figure 3), tryparedoxin, or alkyl hydroperoxide reductase [179,185], In the reduced state, Prdxl, II, and IV form decamers or dodecamers such as PrdxIII [186], Reduced decamers show efficient peroxidase activity and, depending on other posttranslational modifications, form high molecular weight oligomers associated with cell cycle checkpoints, chaperones, and various intracellular processes [187-189], The "atypical 2-Cys" Prdxs (Prdx5) are monomers forming intramolecular disulfide since both CysR and Cysp are within the same molecule; their reduction is achieved by Trx. The "atypical 2-Cys" Prdxs can form dimers independently of the redox state [179]. The "1-Cys" Prdxs (Prdx6) contains only Cysp in the N-terminus [190]. The resolving electron donor thiol can be glutathione, allowing the formation of a mixed disulfide, while the second donor thiol enables the reduction of the formed disulfide bonding. Ascorbate, lipoic acid, and cyclophilin, but most commonly GSH, can serve as electron donors for disulfide reduction [179,185,191], Prdx6 reduces phospholipid hydroperoxides using GSH, and also the GST Pl-1 class showed the ability to act as phospholipase A2 [192], Hyperoxidation, formation of RSO2H or RSO3H, and phosphorylation regulate the activity of Prdxs [181], The "1-Cys" Prdxs are resistant to hyperoxidation. Hyperoxidation can be repaired by sulfiredoxin, but not in human Prdx6 [193], Molecules 2023, 2», 1447 12 of 22 6. Glutathione-S-Transferases (GST) GSTs belong to the Phase II biotransformation enzymes catalyzing the GSH-mediated peroxide reduction [194] and conjugation of GSH with a variety of reactive electrophiles, most commonly generated by cytochrome P450 metabolism [195]. GSTs expressed ubiquitously, but tissue-specific distribution is probably an adaptive response against endo- and exogenous metabolites [196]. GSTs comprise two distinct superfamilies, membrane-bound microsomal and soluble cytosolic. In humans, cytosolic GSTs are encoded by 16 genes, while the microsomal, at least by six genes, in addition to significant genetic polymorphisms [197]. According to the degree of sequence identity and localization, the cytosolic GSTs (cGSTs) are divided into alpha, mu, pi, omega, theta, delta, sigma, and zeta (A, M, P, O, T, D, S, Z) classes. Mitochondrial GSTs (mGSTs) are divided into A, M, P, and kappa (K) classes. A novel superfamily designated MAPEG (Membrane Associated Proteins in Eicosanoid and Glutathione metabolism) includes members of widespread origin with diversified biological functions. Members of this family are leukotriene C-4 synthase, 5-lipoxygenase activating protein, prostaglandin E synthase, and microsomal glutathione S-transferases (MGST) 1,2 and 3 [198,199]. Due to polymorphisms, gene duplication, and genetic recombination, GSTs have multiple isoenzymes with overlapping substrate specificity and diversity [200]. In humans, the highest cytosolic GST activity level is present in the liver, whereas the kidney, lung, and intestine show lower activity levels than that of the liver at 22, 66, and 63%, respectively [201]. Intracellularly, some specific GST activities also were detected in the plasma membrane, outer mitochondrial membrane, and nucleus [198]. In mammals, GSTs exist as homodimers with analogous tertiary structures [202]. All GSTs have a basic protein fold comprising two subunits with C-terminal and N-terminal domains. The N-terminal domain includes a thioredoxin-like fold, |3-2, and protein chaperones. Antioxid. Redox Signal. 2011,15, 781-794. 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Xue, M.; Shafie, A.; Qaiser, T; Rajpoot, N.M.; Kaltsas, G.; James, S.; Gopalakrishnan, K.; Fisk, A.; Dimitriadis, G.K.; Grammatopou-los, D.K.; et al. Glyoxalase 1 copy number variation in patients with well differentiated gastro-entero-pancreatic neuroendocrine tumours (GEP-NET). Oncotarget 2017, 8, 76961-76973. [PubMed] Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. 5 OXIDAČNÝ STRES A OPIOIDY Viaceré štúdie poukazujú na koreláciu medzi vznikom oxidačného stresu a jeho následnými komplikáciami v súvislosti s liečbou opioidmi. Najviac podozrivým liečivom s týmto vedľajším účinkom sa javí byť morfin. Tento efekt je pravdepodobný aj pri semisyntetických molekulách odvodených od morfínu, ktoré sa pri odbúravaní v organizme menia na morfín alebo jeho metabolity. Štúdie iných opioidov zamerané sa nadmernú produkciu voľných radikálov doposiaľ neboli uskutočnené v dostatočnom rozsahu. V zásade genéza tvorby reaktívnych foriem kyslíka a dusíka v spojitosti s dlhodobým užívaním morfínu môže prebiehať dvoma možnými biochemickými cestami. Sú to reakcie, ktoré nasledujú po aktivácii enzýmu NO-syntázy alebo po aktivácii enzýmu fosfolypázy D2. Tieto cesty môžu v organizme prebiehať súčasne (40). Morphin e 1 ORs NOS PID7 NO Ca 2+ O NO O" (perŕwínítrite) MrtSOD NADPH oxidase r- Oj NO Obrázok č.5: Vzťah medzi morfínom a tvorbou voľných radikálov. Diagram schematicky znázorňuje predpokladané dráhy, ktorými môžu opioidné receptory aktivované morfínom indukovať oxidačný stres v cieľových bunkách. Skratky: ORs - Opioidné receptory, ONOO-- peroxynitrit (peroxynitritový anión), PLD2 - foslfolipázaD2, MnSOD - Superoxiddizmutáza (Mn-kofaktor) (Zdroj: spracované podľa Škrabalova, 2013). 90 Štúdie, ktoré skúmali problematiku krátkodobého a dlhodobého pôsobenia morfínu poukazujú na signifikantný pokles hladín glutatiónu v mozgovom tkanive a v pečeni hlodavcov, ako aj v mozgovom tkanive ľudí. Zároveň bolo zistené, že chronická liečba morfínom viedla k zníženiu aktivít antioxidačných enzýmov superoxiddizmutázy, katalázy, glutatión peroxidázy a ďalších enzýmov zahrnutých do antioxidačnej obrany (40, 41). Na oslabení antioxidačnej aktivity u jedincov zaťažených opioidmi sa podieľa veľa ďalších vnútorných, ako aj vonkajších faktorov. Tento efekt závisí od doby trvania liečby, množstva podávaného morfínu, od možných interakcií s inými liečivami, ako aj od zmien v metabolizme pri rôznych ochoreniach. Vo všeobecnosti sa nadmerná tvorba reaktívnych foriem kyslíka a dusíka a súčasne ich znížená eliminácia antioxidačnými molekulami, spája so vznikom oxidačného stresu (42). Tento efekt bol zaznamenaný v rôznych predklinických a klinických štúdiách, a to pri krátkodobom ako aj dlhodobom podávaní morfínu. ECM degradation Obrázok č.6: Vplyv morfínu na rozličné endogénne procesy. ORs - Opioidné receptory, ONOO- peroxynitrit (peroxynitritový anión), PLD2 - fosfolipáza D2, MnSOD -Superoxiddizmutáza (Mn-kofaktor), VEGF - Vascular endothelial growth factor (rastový faktor cievneho endotelu), EGF - Epidermal growth factor (rastový faktor epidermy), NK - c-Jun N-terminal kinase, MMPs - matrixové metaloproteinázy, BAX - Bcl-2-like protein 4, BIM - Bcl-2-like protein 11, BCL2 - B-cell lymphoma 2, FAS - Fas receptor (Zdroj: Gach, 2011). 91 Štúdia sledujúca oxidačné poškodenie ciev preukázala tvorbu reaktívnych foriem kyslíka po dlhodobom podávaní morfínu v endotelových bunkách laboratórnych zvierat. Tento efekt bol neskôr preukázaný aj na endotelových bunkách ľudí. Vysvetlením tohto javu môže byť znížená koncentrácia oxidu dusnatého, zhoršená vazodilatácia, ako aj následný rozvoj aterosklerózy (43). Vplyvom pôsobenia morfínu bolo zaznamenané zvýšenie koncentrácie peroxidu vodíka v prefrontálnom kortexe a v striatum potkanov. Zároveň boli v hipokampe a v pečeni namerané zvýšené koncentrácie produktov lipidovej peroxidácie (44). Zvýšená tvorba voľných kyslíkových radikálov, a to najmä superoxidu, bola preukázaná aj v tkanivových makrofágoch. Naproti tomu výsledky experimentálnych štúdií sledujúcich bunkové línie ľudského neuroblastómu SH-SY5Y po podávaní morfínu nepreukázali jeho prooxidatívny efekt a ani urýchlenie apoptózy týchto buniek. Tento dôkaz môže svedčiť pre zvýšenú rezistenciu nádorového tkaniva voči tvorbe radikálov a následnému poškodeniu (45). Dôležitou reaktívnou molekulou zapojenou do oxidačných procesov iniciovaných morfínom je peroxynitrit, ktorý sa tvorí v reakcii superoxidového aniónového radikálu s oxidom dusnatým. Tieto molekuly slúžia ako signálne molekuly nielen pri rozvoji oxidačného stresu, ale sú zároveň zahrnuté do patogenézy vzniku hyperalgézie ako aj opiátovej tolerancie. Tieto zistenia boli potvrdené pri inhibícii syntézy NO a superoxidu (46). Medzi hlavné enzýmy, ktorých aktivitou sa tvoria NO a superoxid sú syntáza oxidu dusnatého a enzýmy rodiny NOX (NADPH-oxidázy). Zníženie tvorby radikálov vedie k zníženiu pravdepodobnosti vytvorenia morfínom indukovanej antinociceptívnej tolerancie. Tento efekt bol dokázaný na geneticky modifikovaných laboratórnych myšiach s deficienciou NOS, u ktorých po dlhodobom podávaní morfínu nedošlo k rozvoju morfínom indukovanej antinociceptívnej tolerancie. Morfínom indukovaná aktivácia NADPH oxidázy, ktorá následne katalyzuj e tvorbu superoxidu a aktivuje makrofágy, je jedným z viacerých príkladov prepojenia účinku morfínu a jeho vplyvu na imunitný systém. Rovnako dôležité sú neuroimunitné procesy aktivované zvýšenou produkciou prozápalových cytokínov (TNF-a, IL-ip, IL6). Táto signálna interakcia je sprostredkovaná cez u-opioidný receptor, čím následne dochádza ku aktivácii fosfolipázy D a k následnej kaskáde biochemických dejov, ktoré vedú k zvýšeniu intracelulárneho Ca2+. Pri zvýšenom podávaní morfínu bola zistená zvýšená expresia nikotínamidadeníndinukleotidfosfát (NMDA) receptorov. Tento jav v klinickej praxi môže prispievať k vytvoreniu morfínovej závislosti a tolerancie. K zníženiu glukuronidácie morfínu enzýmom UDP-glukuronozyltransferázou (UGT), môže prispieť 92 obmedzený metabolizmus glukózy vplyvom morfínu ako aj samotné ochorenie. Táto metabolizácia prebieha v pečeni. Je však nutné podotknúť, že UGT má viaceré genetické polymorfizmy, ktoré vykazujú rôznu aktivitu (47). Obrázok č.7: Metabolická degradácia morfínu (Zdroj: De Gregoři 2012). Niektoré štúdie potvrdili, že opiáty ovplyvňujú metabolizmus glukózy a hladiny hormónov regulujúcich glukózu (48). Napríklad pri nádorových ochoreniach sa kyselina glukurónová tvorí v pečeni viac z glykogénu alebo aj z glykogénnych aminokyselín než z glukózy. Preto látky, ktoré majú byť metabolizované UGT spôsobujú pokles glykogénu. V štúdii Favaro a kol. dokázali, že pri nádorových ochoreniach nedochádza k mobilizácii glykogénu (49). Bezprostredne z uvedeného môže vyplývať niekoľko skutočností, ktoré významne zasahujú a menia charakter redoxných procesov: 1. Obmedzená aktivita UGT kvôli nedostatku kyseliny glukurónovej a s tým aj metabolizmus podávaných liečiv. 2. Ovplyvnenie redoxného stavu obmedzením najväčšieho zdroja tvorby NADPH v bunke -pentozafosfátového cyklu a to: glukóza-6-fosfátdehydrogenázy (G6PDH). Je známe, že podávanie morfínu vedie k deaktivácii G6PDH. Dochádza k tomu vytváraním konjugátov morfín-G6PDH (50). Práve preto sa tento druhý bod zdá byť závažnejší než nedostatok kys. \ MORPHINE Morphine - 6- glucuronide Morphine -3- glucuronide 93 glukurónovej, či predtým spomínaná alterácia aktivity polymorfných foriem UGT, pretože morfín môže byť metabolizovaný alternatívne a to dvojako: A) morfín+ NADP- morfín dehydrogenáza—^ NADPH + morfinón morfinón + NADH morfinón reduktáza-^ NAD++ hydromorfón hydromorfón + NADPH morfinón dehydrogenáza ^ NADP+ + dihydromorfón NADPH je pritom vyžadované pre aktivitu: 1. NADPH-oxidáz, avšak nakoľko je ich aktivita výrazná najmä u buniek imunitného systému a tie využívajú k jeho tvorbe glutamín, nepredpokladáme preto obmedzenie produkcie superoxidu. 2. syntázy oxidu dusnatého (NOS). Dostatok kofaktorov a substrátov značne ovplyvňuje aktivitu všetkých troch izoforiem NOS. V prípade nedostatku NADPH, tetrahydrobiopterínu, či aj substrátu arginínu (alebo dokonca aj jeho chýbaniu) dochádza k tzv. odpojeniu aktivity NOS, pričom sa tvorí súčasne NO aj superoxid, ktoré reagujú za vzniku peroxynitritu (51). Pterín pôsobí ako účinný vychytávač 02"», čím predchádza rýchlej reakcii 02"» s NO a tvorbe ONO2" a uvoľňuje ho vo forme H2O2. Jedine v prípade nedostatku hemu NOS nie sú schopné viazať tetrahydrobiopterín a nekatalyzujú tvorbu NO (52). 3. Glutatiónreduktázy, ktorej účinkom dochádza prostredníctvom NADPH k spätnej redukcii oxidovaného glutatiónu (GSSG) na redukovaný (GSH). B) cytochrómami: Morfín, oxymorfín aj hydromorfón môžu byť metabolizované N-demetyláciou enzýmami cytochrómu P450 (CYP3A4 a CYP2C8). Tento reakčný mechanizmus je taktiež závislý na NADPH. Z neho preberá elektróny na vytvorenie redukovaného hemu. Redukovaný hem umožní naviazanie 02 a oxidáciu substrátu vložením jedného kyslíka do substrátu a druhého do molekuly vody. Reakciu je možné znázorniť takto: P450red + RH + 02 -► P450ox + ROH + H20 94 Perferylový komplex [Fe = O] , ktorý sa tvorí na heme prijatím elektrónu je schopný namiesto OH skupiny naviazať iba H čím sa tvoria aj alkylové radikály. Radikály poškodzujú bunkové štruktúry reakciami s proteínmi za vzniku stabilných adduktov. Okrem toho sa ich aktivitou ako vedľajší produkt môže tvoriť singletový kyslík, vrámci reakcií sú však schopné redukovať molekulárny kyslík na superoxid a/alebo H2O2 (5). Výrazný vplyv tu zohráva aj unikátna vlastnosť enzýmu syntázy oxidu dusnatého (NOS). Enzým je podobný cytochrómu P450 a katalyzuje iné oxidačné metabolické reakcie, v ktorých sa taktiež tvoria radikálové intermediáty. Mechanizmus oxidácie látok je možné vysvetliť mechanizmom cytochrómu P450. Cytochróm P450[Fe5+ = 0]3+ odtrhnutím vodíka zo substrátu vytvorí cytochróm P450[Fe4+ - OH]3+ a zodpovedajúci radikál (53). Skutočnosť, že NOS metabolizuje a ovplyvňuje účinnosť a toleranciu k morfínu naznačujú aj niektoré štúdie, napríklad už dostatkom arginínu (54). Ďalšie štúdie poukázali na antinociceptívny účinok morfínu, ktorý bol zaznamenaný pri aplikácii L-arginínu. Naproti tomu podávanie D-arginínu tento efekt nemalo. Čo jednoznačne naznačuje výrazný vplyv NOS na metabolizmus a účinnosť morfínu, ako aj tvorbu reaktívnych častíc, nakoľko NOS má obmedzenú substrátovú špecificitu a k syntéze NO nedokáže D-arginín využiť (53, 55). 5.1 Štúdia Opioid-Redox Study Viaceré dôkazy z experimentálnych štúdií, ako aj výsledky niektorých klinických štúdií poukazujú na úlohu opioidov pri vzniku oxidačného stresu. Tento jav sa rovnako predpokladá u pacientov s chronickou bolesťou, odkázaných na dlhodobé užívanie silných opioidov, ktoré sú pilierom liečby neuropatickej bolesti. Štúdie v tejto skupine pacientov absentujú a to najmä pre vysoko komplexnú problematiku spojenú s polymorbiditou daných pacientov s rôznymi pridruženými ochoreniami, odlišnou dĺžkou liečby analgetikami, individuálnym dávkovaním analgetík, ako aj liekovými interakciami, farmakokinetikou a farmakodynamikou celého spektra liečiv, ktoré pacienti užívajú. Z dôvodu nejasných dôkazov o intenzite oxidačného stresu u pacientov s chronickou bolesťou liečených opioidmi pre silnú bolesť náš lekársko-vedecký tím realizuje prospektívnu observačnú mul ti centrickú klinickú štúdiu pod názvom Opioid-Redox Study. Tento projekt je realizovaný v spolupráci s Algeziologickou klinikou SZU Fakultnej nemocnice s poliklinikou F. D. Roosevelta v Banskej Bystrici, Ambulanciou bolesti Algmed v Košiciach, 95 Ústavom lekárskej biochémie Univerzity Pavla Jozefa Šafárika v Košiciach a Východoslovenským ústavom srdcových a cievnych chorôb a.s. v Košiciach. Štúdia bola zaregistrovaná v medzinárodnej databáze klinických štúdií „clinicaltrials.gov" pod registračným číslom NCT03105232. Cieľom štúdie je monitorovať klinický stav pacientov, zmeny aktivít antioxidačných enzýmov a redoxných kapacít po začatí liečby opioidmi pre silnú bolesť, ako aj v priebehu ich užívania. Tento projekt aktuálne prebieha vo fáze náboru pacientov do klinickej štúdie. Po splnení inklúznych kritérií a zaradení do štúdie sú pacienti rozdelení do 4 skupín: Chronická bolesť Morfín, Hydromorfon, Oxykodón Buprenorfín Fentanyl Tapentadol Obrázok č.8: Flow diagram klinickej štúdie Opioid-Redox Study. Hodnotia sa nepriame markery oxidačného stresu, medzi ktoré patria: aktivita glutatión peroxidázy, glutatión reduktázy, katalázy, superoxiddizmutázy a plazmatických hladín glutatiónu. Monitorované sú klinické parametre ako doterajšia spotreba analgetík, vyšetrenie zamerané na typ bolesti (nociceptívna vs. neuropatická) hodnotením senzitívnych dotazníkov PainDetect, DN4, a LANSS Pain škály. Jednotlivé merania sa realizujú v intervaloch: pred začatím užívania opiátov, po 6 mesiacoch a po 12 mesiacoch danej opiátovej liečby. Niektoré štúdie zaoberajúce sa problematikou dlhodobého užívania opiátových analgetík a následnými zmenami oxidačno-redukčných dejov, poukázali na nižšiu úroveň nežiadúcich oxidačných účinkov morfínu v porovnaní s ekvivalentnými dávkami iných opioidov (47, 56). Pilotné výsledky štúdie Opioid-Redox Study poukazujú na rovnakú intenzitu prebiehajúcich antioxidačných reakcií u všetkých skupín pacientov, ktorí sú na dlhodobej opiátovej liečbe. Bol zaznamenaný signifikantný vzostup plazmatickej aktivity enzýmov - glutatión peroxidázy, glutatión reduktázy a signifikantné zvýšenie plazmatickej koncentrácie glutatiónu. Zároveň došlo u týchto sledovaných skupín k signifikantnému poklesu aktivít superoxid dizmutázy, v porovnaní s kontrolnou skupinou zdravých jedincov, čo poukazuje na 96 zvýšenú úroveň prebiehajúcich antioxidačných reakcií u pacientov s chronickou bolesťou po 6 mesiacoch na opiátovej liečbe. Tieto údaje naznačujú možnú prítomnosť oxidačného stresu v opiátovej skupine. Doterajšie závery tejto práce boli prezentované na medzinárodnej konferencii: • Simonová J, Vašková J, Ogurčáková D, Kočan L. Martuliak I, Rapčan R, Simon R. Antioxidant capacities of patients with chronic pain after strong opioid treatment. Pain in Europe XI Valencia, European Pain Federation EFIC 2019. Práce súvisiace s problematikou vplyvu opiátov na rozvoj oxidačného stresu boli publikované v recenzovaných medicínskych časopisoch a časopisoch WOS: • Ogurčáková D, Kočan L, Simonová J, Martuliak I, Sabol F, Vašková Janka. Oxidative stress in patients receiving long-term opioid therapy. Laboratórna diagnostika: recenzovaný časopis pre pracovníkov diagnostických laboratórií. ISSN 1335-2644. - Roč. 24, č. 1 (2019), s. 57-61. • Kočan L, Martuliak I, Ogurčáková D, Vašková J, Kočanová H. Oxidačný stres u pacientov dlhodobo liečených opiátmi. Paliatívna medicína a liečba bolesti. - ISSN 133 7-9917. - Roč. 8, supl. 2 (2015), s. 23. • Ogurčáková D, Kočan L, Simonová J, Martuliak I, Sabol F, Vašková Janka. Plasma antioxidant status in patients undergoing long-term opioid treatment. Medical Science. ISSN 2321-7367. - Roč. 26, č. 124 (2022). s 1-7. DOI: 10.54905/disssi/v26i 124/ms217e2319. 97 MEDICAL SCIENCE I RESEARCH ARTICLE Medical Science pISSN 2321-7359; elSSN 2321-7367 To Cite: Ogurcakova D, Kočaii L, Šimonova J, M.irlulj.ik I, Sabol F, Vašková J. Plasma antioxidant status in patients undergoing long-term opioid treatment. Medical Science, 2022, 26, ms217e2319. Author affliations 'Pain management clinic Algmed, Rastislavova 45, 04001 Košice, Slovak Republic -Department of Medical and Clinical Biochemistry, Faculty of Medicine, Pavol Jozef Safárik University in Košice, Trieda SNP 1, 040 11 Košice, Slovak Republic 'Clinic of Anaesthesiology and Intensive Care Medicine, Kast Slovak Institute of Cardiovascular Disease, Ondavská 8,040 11 Košice, Slovak Republic 41" Clinic of Anaesthesioiogv and Intensive Care Medicine, Louis Pasteur University Hospital, SNP 1, 040 11 Košice, Slovak Republic ^Department of Algeziology, F.D. Roosevelt Teaching Hospital with Policlinic Banská Bystrica and Slovak Medical University in Banská Bystrica, nám. Ludvika Svobodu 8, 97401 Banská Bystrica, Slovak Republic 'Deparlment of Cardiac Surgerv, Last Slovak Institute of Cardiovascular Disease, Ondavská 8, 040 11 Košice, Slovak Republic 'Corresponding author Janka Vašková, assoc. prof. Dr. PhD. Department of Medical and Clinical Biochemistry. Faculty of Medicine, Pavol Jozef Safárik University in Košice, Tr. SNP 1, 04066 Košice, Slovak Republic Lm.iil; janka.vaskowv upjs.sk Peer-Review History Received: 26 May 2022 Reviewed & Revised: 27/May/2022 to 09/June/2022 Accepted: 09 June 2022 Published: 11 June 2022 Peer-review Method External peer-review was done through double-blind method. URL: https://www.discuveryjournals.org/medicalscience This work is licensed under a Creative Commons Attribution 4.0 International License, DISCOVERY SCIENTIFIC SOCIETY Copyright © 2022 Discovery Scientific Society- Plasma antioxidant status in patients undergoing long-term opioid treatment Ogurčáková Daniela1-2, Kočan Ladislav3, Simonová Jana4, Martuliak Igor5, Sabol František6, Vašková Janka2* ABSTRACT Background: Opioid treatment is now an integral part of pharmacotherapy for severe chronic malignant and non-malignant pain. Currently, there is a sufficient selection of opioids to allow individualized pain treatment. Several experimental studies have confirmed the effect of opioids on oxidative stress. The aim of this work is to determine the presence of redox changes occurring as a result of long-term opioid use in patients with chronic pain. Results: Six months of opioid use for severe pain was evaluated in 37 patients. Patients formed three groups depending opioid treatment (oxycodone, fentanyl and tapentadol) and were compared with 42 healthy probands. Compared to control, activities of superoxide dismutase were decreased, while those of glutathione peroxidase and glutathione reductase were significantly increased in all groups. Together with lowered levels of reduced glutathione, this indicated conditions of oxidative stress. There were no differences between treatment groups. Conclusion: It is necessary to know the risks of side effects and provide patients with possible solutions. At this stage and with this number of subjects, we can conclude that neither the form of administration nor the type of opioid has any effect on reducing oxidative stress from opioid metabolism in the treatment of severe pain. Keywords: antioxidant enzymes, pain, glutathione, opioids, oxidative stress 1. INTRODUCTION Chronic pain and non-malignant pain is a widespread serious public health problem (Breivik et al., 2006; Ripamonti, 2012). There are no clear epidemiological studies available in the European Union; however, approximately 50% of adults suffer from one or more chronic pain with an incidence of moderate to severe pain in the European population at 20%. 70% of patients are in active age, 11% of patients are untreated, and 50% do not have enough pain treatment. The prevalence is higher in women and the number of patient's increases with age, reaching 80% in geriatric patients. The economic costs, namely indirect costs dLie to incapacity for work, are not negligible. The most common chronic pains such as back pain, osteoarthritis, headache, and neuralgia are often considered to be a normal part of life. The treatment of chronic pain is the elimination of pain and restoration of all Medical Science, 26, ms217e2319 (2022) lof 7 MEDICAL SCIENCE I RESEARCH ARTICLE_ functions (physical, mental, social); in the most optimal case, this includes the possibility of returning to work (Breivik et al., 2006; 2013). The choice and management of analgesic treatment is based on patient data regarding the intensity and nature of the pain and on the specific clinical condition. It does not determine the origin of the pain (malignant, non-malignant), but in chronic pain: the procedure follows a "bottom-up" approach (step up). In intense acute pain, parenteral administration of an analgesic, possibly also an opioid (in cases of angina pectoris, heart attack, renal and gallbladder colic) is appropriate; otherwise non-invasive administration of analgesics (per os, transdermal, per rectum) is preferred. Regarding time, analgesics with a rapid onset of action are the most advantageous in acute pain; in chronic pain, analgesics are administered "on an hourly basis" and thus the development of pain is prevented. Opioid analgesics are essential for pain management (Gilson et al., 2011) due to efficiency and safety treatment under competent physicians. The biopsychosocial status in non-terminal patients with chronic pain should be considered by physician to set up a treatment plan with patient motivation to reach functional goals (Von Korff et al., 2011; Kalso et al., 2004). Morphine is considered to be the gold standard of opioid treatment, with the properties of other opioids derived from it. Many side effects of opioid treatment are under thorough investigation. Recent findings suggest that long-term opioid treatment may contribute to oxidative stress, which is a serious pathological problem due to its key role in the pathogenesis of many diseases (Cacciapuoti, 2016). Recent studies have also revealed new roles for oxidative stress or reduced antioxidant activities relevant to mitochondria functions behind the development of a migrains (Ferroni et al, 2018). Clinical trials focusing on the redox state of patients taking opioids for chronic pain are still lacking. Due to unclear evidence of oxidative stress intensity in chronic pain patients dosed with opioids for severe pain we have started a prospective multicentre observational study. The aim of the study was to monitor the clinical condition of patients, as well as changes in the activities of antioxidant enzymes after starting opioid treatment for severe pain and during their use. 2. MATERIALS AND METHODS The study was carried out in co-operation of three pain treatment centres, Pain management clinic Algmed in Košice, Department of Algesiology, F.D. Roosevelt Hospital in Banská Bystrica, and the East Slovak Institute of Cardiovascular Diseases in Košice. The Ethic Committee of Faculty of Medicine Pavol Jozef Šafárik University in Košice no. IN/2017, Ethic Committee of Slovak Medical University in Banská Bystrica 28/11/2016, and Ethic Committee of East Slovak Institute of Cardiovascular Diseases in Košice no. 1/2019/VUSCH/EK approved the study. The study was registered in the International Database U.S. National Institutes of Health ClinicalTrials.gov under the number NCT03105232. Patients recruitment started august 2020 and was completed December 2021. The course of the research was explained to potential study participants and, after signing informed consent, followed up with a pain specialist. Screening of parameters was carried out, namely: consumption of analgesics, pain type examination (nociceptive vs. neuropathic - filling out the questionnaires for Pain Detect, DN4, and numeric pain scale), and demographic data (weight, height, age). The inclusion criteria of patients were: non-malignant pain, no previous opioid treatment for visceral, neuropathic, or nociceptive, pain and age over 18 years. Patients with oncological conditions were excluded. Enrolled participants were divided into 4 groups: group 1 - control group (C), healthy individuals (42); group 2 (O) - patients with chronic pain using morphine, hydromorphone, oxycodone, or buprenorphine (14); group 3 (F) - patients with chronic pain receiving transdermal fentanyl patch (12); group 4 (T) - patients with chronic pain using tapentadol (11). The second examination followed six months from the start of opioid use for severe pain. During the examination, blood for biochemical analysis was collected, patients completed a Pain Detect questionnaires, DN4, and Lanss Pain Scale, and clinical parameters were measured. Bicinchoninic acid was used for blood plasma protein determination. Glutathione peroxidase, glutathione reductase and glutathione-S-transferase activities were determined according to the procedures given by the kit manufacturer (Sigma-Aldrich, Germany). Superoxide dismutase activities were set according to the SOD-Assay KIT-WST (Fluka, Japan). The concentration of reduced glutathione (GSH) was determined by the method originally described by Floreani et al. (1997). Descriptive statistics were used to characterise groups of patients. A T-test was used to compare values within groups against the corresponding control. Tntragroup differences at two sampling times were determined by one-way analysis of variance followed by Tukey-post hoc test. Intergroup differences within parameters were detected by a Mann-Whitney test. Differences were considered significant at p < 0.05, p < 0.01, p < 0.001. Medical Science, 26, ms217e2319 (2022) 2 of 7 MEDICAL SCIENCE I RESEARCH ARTICLE 3. RESULTS AND DISCUSSION After screening the patients who met the conditions and completed both samplings (also after 6 months), 37 patients were included in the study. The measurements of antioxidant parameters in plasma were compared with a group of 42 healthy individuals. Results (Figure 1 and 2) show that SOD activities were decreased in each treatment group when compared to control before the start of therapy and after 6 months regardless of the form of drug administered and the type of opioid. Activities of GPx and GR were increased in comparison to control at both time points. The activities of both GPx and GR enzymes have an even greater tendency to increase 6 months after opioid treatment in all groups. In group 3, there was a statistically significant increase in GPx (p=0.0044) over 6 months (Table 1). Morphine treatment affects antioxidant enzyme activities, as morphine-dependent rhesus macaques have been observed with GPx, SOD increased after 140 days of morphine treatment (Pérez-Casanova et al., 2008). Other studies have shown that these enzyme activities were reduced by morphine (Payabvash et al., 2006; Sumathi et al., 2011; Zhou et al., 2011; Roziski et al., 2013). It becomes obvious that this can be affected by many different factors including dosage, exposure time, and species exposed. The catalytic activity of SOD is the dismutation of the superoxide radical (O2") to hydrogen peroxide. Hydrogen peroxide as well as other hydro peroxides is converted to water or the corresponding alcohols by GPx. Glutathione is co-oxidized in the reaction, and reduced back by GR, thus providing an effective antioxidant response. The increased activities of these two glutathione-related enzymes are confirmation of oxidative stress conditions in patients suffering from pain, and very likely suppression or inactivation of SOD. Moreover, O2" as well as peroxynitrite anion (ONO2), are known as pro-noci cep five species (Salvemini and Newmann, 2009; 2010). Under conditions of oxidative stress, inflammation contributes even more significantly to raising the concentration of O2" through several sources. In the respiratory chain within mitochondria, these include lipoxygenase, cyclooxygenase, NOX enzyme induction, and concomitantly the formation of the ONO2" as a result of thiol oxidation in xanthine dehydrogenase, and uncoupling of NOS activity (Vašková et al., 2016). 16 14 12 SOD(ukat/mg GPx(ukat/mg GR(nkat/mg GSH(nmol/mg GST(ukat/mg prot) prot) prot) prot) prot) ■ Control bO bF P Figure 1 Antioxidant enzyme activities and reduced glutathione concentrations in patients treated with opioids for severe pain compared to healthy subjects at the beginning of treatment period. Statistical significance at *p < 0.05 a ***p < 0.001 Reactive oxygen species (ROS) formation has been observed even at low doses of morphine in vascular endothelial cells (mouse and human) (Hsiao et al., 2009). Macrophages have also been found to be morphine-induced generating O2" (Bhat et al, 2004). Other morphine-induced sources are activated nitric oxide synthase (NOS) and NADPH oxidase. Activation of NOS leads to increase NO production and subsequent SOD nitration. Inactivation of SOD leads to the formation of ONO2- in a reaction between Or and NO. Although O2 is an initiator of the formation of other ROS, ONO2 together with NO depletion have deleterious effects on tissues and their functions (Vašková et al., 2016). All these ROS are involved in the pain sensitisation, opiate-induced hyperalgesia and antinociceptive tolerance (Salvemini et al., 2009). Medical Science, 26, ms217e2319 (2022) 3 of 7 MEDICAL SCIENCE I RESEARCH ARTICLE n | 15 | I 10 ill .1 III, ..I: SOD(nkat/mg GPx(nkat/mg GPx{nkat/mg GSH(nmol/mg GST(nkat/mg prot) prot) prot) prot) prot) Figure 2 Antioxidant enzyme activities and reduced glutathione concentrations in patients treated with opioids for severe pain compared to healthy subjects 6 months after starting therapy. Statistical significance at **p < 0.01 and ***p < 0.001. SOD is inactivated in vivo through nitration with NO-derived oxidants (ONO2) and hydroxylation (hydroxyl radical) (Janssen-Heininger et al., 2005). Salvemini et al., (2011) reported MnSOD inactivation as an essential element for increased production of O2" and ONO2- in nociceptive signalling from the results of several studies of their research group. Inactivation of NOS or inhibition of nitration and inactivation of SOD made it possible to prevent the formation of morphine-induced antinociceptive tolerance (Muscoli et al., 2007). It was found that hydrogen peroxide is capable of SOD inactivation, yet too slow to be the cause of inactivation under physiological conditions (Escobar et al, 1996). However, relatively higher concentrations of hydrogen peroxides (and hydroperoxides) are capable of substrate peroxidase inactivation (Olorunniji et al., 2009), which was not confirmed by the results of our study. Increased activities of GPx, synergistically acting GR together with lowered concentrations of GSH (especially 6 months after starting therapy) only support the response to oxidative stress conditions (Figure 1 and 2). Levels of GSH were significantly higher when compared with control (p < 0,001) in group treated with oxycodone, hydromorphone at the beginning, and unchanged in group with fentanyl, they markedly decreased 6 months after starting therapy (p < 0,001). Table 1 Further description of the results of the antioxidant parameters in patients treated with opioids for severe pain compared to healthy subjects before at the beginning (0) and 6 months of treatment (6). Significance at b <0.01, c<0.05 Group SOD GPx GR GSH GST (ukat/mg prot) (ukat/mg prot) (ukat/mg prot) (nmol/mg prot) (ukat/mg prot) med (min-max) med (min-max) med (min-max) med (min-max) med (min-max) O 0 0.24 (0.16-0.99) 6.61 (0.99-11.6) 14.06 (1.37-27.29) 9.76 (0.78-12.23) 3.35 (0.97-4.53) c 0.24 5.60 9.18 1.86 0.55 (> (0.22-0.61) (0.94-29.11) (4.65-13.12) (1.70-5.21) (0.20-2.74) F 0 0.22 (0.19-1.21) 9.98 (1.09-26.94)b 9.23 (3.32-19.07) 2.51 (1.01-4.38) 3.08 (0.15-10.36) 0.27 7.00 9.75 2.51 1.55 6 (0.19-1.39) (1.12-29.11)" (1.83-10.39) (0.56-3.96) (0.60-9.01) T 0 0.23 6.72 12.52 2.39 4.19 (0.17-1.21) (2.23-21.04) (2.53-22.58) (0.76-4.83) (0.18-12.23)= 0.24 5.45 22.32 1.33 0.44 6 (0.21-0.63) (0.69-6.35) (11.7-28.34) (1.19-1.97) (0.19-0.56)» Medical Science, 26, ms217e2319 (2022) 4 of 7 MEDICAL SCIENCE I RESEARCH ARTICLE Unlike other opioids, morphine is more associated with the induction of oxidative stress either by the formation of ROS or reduction the activity of antioxidants (Skrabalova et al., 2013; Zahmatkesh et al. 2017). Several studies demonstrated that both acute and chronic morphine exposure can lead to significant reductions in GSH levels in rodent and human brains, serum and liver (Abdel-Zaher et al., 2010; Cemek et al., 2011; Guzman et al., 2006; Mannelli et al., 2009; Ozmen et al., 2007; Payabvash et al., 2006; Skoulis et al., 1989; Sumathi et al., 2011; Todaka et al., 2005). Methadone and buprenorphine treatment showed similar results (Leventelis, et al., 2019). In addition to the examined function of glutathione within this study, this molecule is much more widely involved in redox reactions, e.g. reacting directly with Or, H2O2 and NO, participating in disulphide interchange, amino acid transport into the cells in the y-glutamyl cycle and conjugation with electrophilic compounds catalysed by GST. A significant increase in GST activities was found in every group at the beginning of treatment (Figure 1). After 6 months, there was a decrease in GST activity in group 2 with no difference from the control (Figure 2). Yet, in the group treated with tapentadol, activities decreased in comparison with control, and there was the significant difference between times of sampling (p=0.0019) in this group (Table 1). Myers et al., (2010) found a six-fold increase in protein level and expression of liver GST isoenzyme after administration of oxycodone to rats for 8 days. However, in the case of this study, the first measurements were not made after the short-term effect of the opioids, but before their action. Observed increases in activities of GST may be due to the response to the oxidative stress conditions in patients. In 6 month of opioid administration, there was decrease in GST activity, in all three groups, however most in group 4. Comparable effect in long-term morphine use was showed on rat model study (Samarghandian et al., 2014). Although some studies pointed to lower levels of adverse effects in comparison to equivalent doses of opioids such as morphine, the results of our study did not show significant changes between the monitored antioxidant parameters in the three various opioid treatment groups over a period of half a year. In all three groups, a very similar pattern of action was found on antioxidant enzymes, conjugation with glutathione and the effect of reduced glutathione levels. Acknowledgement We would like to thank the Department of Medical and Clinical Biochemistry for the support. Author contributions KL, MI and JV conceived and designed the study. Data collection and measurements were performed by DO, ŠJ and MI. The manuscript was written by DO, JV and SF, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Funding Study was supported by grant VEGA 1/0782/15. 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J Clin Invest 2007; 117: 3530-3539. doi: 10.1172/JCI 32420. 18. Myers AL, Hassan HE, Lee IJ, Eddington ND. Repeated administration of oxycodone modifies the gene expression of several drug metabolising enzymes in the hepatic tissue of male Sprague-Dawley rats, including glutathione S-transferase A-5 (rGSTA5) and CYP3A2. J Pharm Pharmacol 2010; 62: 189-196. doi: 10.1211/jpp.62.02.0006. 19. Olorunniji F, Iniaghe MO, Adebayo JO, Malomo SO, Adediran SA.Mechanism-based inhibition of myeloperoxidase by hydrogen peroxide:enhancement of inactivation rate by organic donor substrates. Open Enzym Inhib J 2009; 2: 28-35. 20. Ozmen I, Naziroglu M, Alici HA, Sahin F, Cengiz M, Eren I. Spinal morphine administration reduces the fatty acid contents in spinal cord and brain by increasing oxidative stress. Neurochem Res 2007; 32: 19-25. doi: 10.1007/sll064-006-9217-5. 21. Payabvash S, Beheshtian A, Salmasi AH, Kiumehr S, Ghahremani MH, Tavangar SM, Sabzevari O, Dehpour AR. 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Targeting peroxynitrite driven nitroxidative stress with synzymes: A novel therapeutic approach in chronic pain management. Life Sci 2010; 86(15-16):604-614. doi: 10.1016/j.Ifs.2009.06.011. 26. Salvemini D, Neumann WL. Peroxynitrite: a strategic linchpin of opioid analgesic tolerance. Trends Pharmacol Sci 2009; 30:194-202. doi: 10.1016/j.tips.2008.12.005. 27. Samarghandian S, Afshari R, Farkhondeh T. Effect of long-term treatment of morphine on enzymes, oxidative stress indices and antioxidant status in male rat liver. Int J Clin Exp Med 2014; 7:1449-1453. PMID: 24995110. 28. Skoulis NP, James RC, Harbison RD, Roberts SM. Depression of hepatic glutathione by opioid analgesic drugs in mice. Toxicol Appl Pharmacol 1989; 99: 139-147. doi: 10.1016/0041-008x(89)90119-1. 29. Skrabalova J, Drastichova Z, Novotný J. Morphine as a Potential Oxidative Stress-Causing Agent. Mini Rev Org Chem 2013; 10: 367-372. doi: 10.2174/1570193X113106660031. 30. Sumathi T, Nathiya VC, Sakthikumar M. 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Protective role of taurine against morphine-induced neurotoxicity in C6 cells via inhibition of oxidative stress. Neurotox Res 2011; 20: 334-342. doi: 10.1007/sl2640-011-9247-x. Medical Science, 26, ms217e2319 (2022) 104 6 ĎALŠIE PRÁCE SPOJENÉ S TEMATIKOU AKÚTNEJ A CHRONICKEJ BOLESTI Rozsiahlou činnosťou, ktorá sa stala predmetom mojej ďalšej medicínskej práce a klinického výskumu je liečba a štúdium akútnej a chronickej bolesti, keďže bolesť predstavuje bežný, avšak rozsiahly medicínsky problém týkajúci sa hospitalizovaných ako aj ambulantných pacientov. 6.1 Problematika bolesti Bolesť je komplexný fenomén, ktorý postihuje milióny ľudí na celom svete a jej zvládanie zostáva hlavnou výzvou pre systémy zdravotnej starostlivosti. Nová revidovaná definícia Medzinárodnej spoločnosti pre štúdium bolesti (IASP) charakterizuje bolesť ako nepríjemný senzorický a emocionálny zážitok, ktorý je spojený so skutočným alebo potenciálnym poškodením tkaniva. Definícia ďalej poukazuje na individuálny aspekt každého jedinca a zdôrazňuje rozdiel medzi nocicepciou a bolesťou, keďže bolesť sa nedá odvodiť len zo samotnej aktivity senzorických neurónov. Tvrdí, že v priebehu života jedinca sa vytvárajú behaviorálne vzorce súvisiace s prežívaním bolesti. Zdôrazňuje ďalej to, že ak pacient vyjadruje svoju bolesť, tak táto informácia má byť rešpektovaná. Verbálny popis bolesti je len jedným z viacerých prejavov (expresie) bolestí a neschopnosť pacienta komunikovať neznamená, že pacient neprežíva pocit bolesti (57). Bolesť je možné definovať podľa rôznych kritérií. Na základe časového faktora trvania bolesti rozlišujeme bolesť akútnu a bolesť chronickú. Časovým medzníkom je trvanie bolesti viac alebo menej ako 3 mesiace. Akútna bolesť je v určitom zmysle chápaná ako eudynia, takzvaná „dobrá bolesť", ktorá je signálom akútneho poškodenia tkaniva a teda má ochranný význam. Avšak táto predstava stráca na význame za okolností, ktoré majú závažný dopad na klinický stav pacienta. Táto situácia nastáva pri jej silnej intenzite, kedy svojimi metabolickými a imunologickými zmenami môže poškodiť zdravie jedinca. Vhodným príkladom je pooperačná bolesť u hospitalizovaných pacientov. Jej mnohostranné negatívne dopady na perspektívu zdravia pacienta sú vyjadrené v tabuľke (58, 59). 105 ASPEKT MORBIDITA oddialenie hojenia vyvolané zvýšenou sympatikotóniou ■f" incidencia insuficiencie anastomóz klinicky "ť incidencia pľúcnych komplikácií, vrátane pneumónií, pri šetrení dýchania vyvolanom bolesťou /f- riziko trombotických komplikácií ~T mortalita pretrvávajúca hyperadrenergná odpoveď s hypertenziou zbytočné utrpenie poruchy spánku retencia moču obmedzená mobilita, dýchanie, 4/ autonómia pacienta strach a obavy zbytočná čiastočná alebo úplná neschopnosť so stratou produktivity práce pomalšia rekonvalescencia normálnych funkcií a životného štýlu ■1- kvalita života počas rekonvalescencie perspektívny predĺžený pobyt na JIS alebo v nemocnici predĺžený pobyt na JIS alebo v nemocnici ■T* riziko komplikácií a nákladov na starostlivosť perspektívny T riziko chronickej bolesti a následných nákladov na starostlivosť dôsledkom nedostatočnej kontroly akútnej bolesti je zlá úroveň zdravotníckej starostlivosti Tabuľka č. 1: Negatívne dopady pooperačnej bolesti (Zdroj Kulichová 2011). Chronická bolesť je patologickou bolesťou s predpokladaným pretrvávaním viac ako 3 až 6 mesiacov, teda doby potrebnej na hojenie tkaniva. Často nemá jednoznačne definovanú príčinu a chápeme ju ako „škodlivú bolesť" - maldýniu. Nekontrolovaná sa stáva zmyslom pacientovho života. Odlíšenie chronickej od akútnej bolesti je založené nielen na časovom faktore jej trvania a symptomatológii (charakter, kvalita bolesti, psychické zmeny a podobne), ale predovšetkým vo vyvíjajúcich sa patofyziologických zmenách nervového systému. Tieto zmeny sú následne sprevádzané typickým "algickým" správaním jedinca. (61, 60). Bez ohľadu na to, či je príčina známa alebo ide o bolesť bez zrejmej organickej podstaty, chronická bolesť nie je symptómom ochorenia, ale stáva sa samostatným ochorením s komplikovanou etiológiou, patofyziológiou a symptomatikou (59, 61). 106 6.2 Projekty manažmentu terapie bolesti v nemocničnom zariadení VÚSCH a.s. Riešením akútnej bolesti v zdravotníckom zariadení Východoslovenskom ústave srdcových a cievnych chorôb a.s. v Košiciach (VÚSCH a.s.), bolo vytvorenie podmienok pre vznik Služby akútnej bolesti - (angl. - Acute Pain Servis - APS), vytvorenie tímu APS a rozdelenie úloh pre členov tímu APS na jednotlivých oddeleniach, ako aj etablácie vypracovaných štandardných postupov liečby pooperačnej bolesti na jednotlivých oddeleniach. V rámci tohto projektu boli zavedené do praxe nové techniky pre liečbu prelomovej bolesti, akými boli periférne nervové blokády, fasciálne blokády a kryoablačné techniky po kardiochirurgických výkonoch, sternotómiach a torakotómiach. Výsledky týchto metód boli sledované v klinickej štúdii VUSCH/POPT1 study NCT03915301 a ďalej prezentované na kongresoch Donovaly 2023, Brno 2023 a publikované v časopise Medical science, indexovanom vo WOS. • Kocan L., Rapčan R, Rapcanova S., Var hol J., Vašková J. Cryoablation of the intercostal nerve after mini-thoracotomy procedures: Pilot prospective interventional clinical study. Medical Science. ISSN 2321-7367. Volume 27, Issue 142, December 2023. DOI: https://doi. org/10.54905/disssi. v2 7i 142. e384ms3258 Problematika chronickej bolesti v zdravotníckom zariadení VÚSCH a.s. bola riešená založením Ambulancie chronickej bolesti v roku 2018, ktorá j e určená prevažne pre pacientov s vysokým kardiovaskulárnym rizikom. Okrem štandardnej komplexnej farmakoterapie akútnej a chronickej bolesti sa zaoberáme intervenčnými výkonmi realizovanými pod ultrazvukovou a skiaskopickou navigáciou. V rámci sledovania klinického stavu pacientov s chronickou ischemickou chorobou dolných končatín (ICHDK) po intervenčných výkonoch zameraných na terapeutické ovplyvnenie lumbálneho sympatikového nervového systému sa realizuje v spolupráci s Technickou Univerzitou v Košiciach prospektívna observačná klinická štúdia: Tevi-LuSy-Study. Štúdia je registrovaná v medzinárodnej databáze „clinicaltrials.gov" pod registračným číslom NCT06111599. Klinická štúdia je finančne podporená grantom: Slovenskej spoločnosti pre štúdium a liečbu bolesti 24-3055/069. 107 ICHDK p roxi má lny uzáver artérií DK distalny uzáver artérií DK kompletný uzáver artérií DK Obrázok č.9: Flow diagram klinickej štúdie Tevi-LuSy-Study. Prvotné výsledky štúdie demonštrujú zvýšenie tkanivovej perfúzie dolných končatín, ktoré boli pôvodne bez priameho prietoku v dôsledku chronickej oklúzie tepien. Zvýšenie perfúzie bolo nepriamo zaznamenané termografickým meraním teplotných zmien v angiozómoch po lumbálnej sympatikovej blokáde. Tieto zistenia naznačujú, že lumbálna sympatiková blokáda môže byť prospešná pre pacientov s ICHDK. Pilotný článok: Thermovision controlled lumbar sympathetic blockade in chronic limb-threatening ischemia treatment - pilot trial, bol publikovaný v karentovom časopise: Vaša -European Journal of Vascular Medicine, Q4, IF 1,8. • Kočan L, Rajťúková V, Rašiová M, Kočanová H, Rapčanová S, Rapčan R, Martuliak I, Hudák R, Rybár D, Vašková J, Hudák M. Thermovision controlled lumbar sympathetic blockade in chronic limb-threatening ischemia treatment - pilot trial. Vasa. 2023 Mar;52(2): 133-135. doi: 10.1024/0301-1526/a001053. PMID: 36872886. 108 133 Letter to the editor i = < = Thermovision controlled lumbar sympathetic blockade in chronic limb-threatening ischemia treatment - pilot trial Ladislav Kočan1,2, Viktoria Rajťúková4 , Maria Rašiová3, Hana Kočanová5, Simona Rapčanová6, Robert Rapčan6, Igor Martuliak7, Radovan Hudák4 , Dušan Rybář1 , Janka Vašková8 , and Marek Hudák3 1 Clinic of Anaesthesiology and Intensive Care Medicine, East Slovak Institute of Cardiovascular Disease, Košice, Slovak Republic 2 Poliklinika Terasa, EuroPainClinics, Košice, Slovak Republic 3 Department of Angiology, East Slovak Institute of Cardiovascular Disease, Košice, Slovak Republic 4 Department of Biomedical Engineering and Measurement, Faculty of Mechanical Engineering, Technical University of Košice, Slovak Republic 5 Clinic of Anaesthesiology and Intensive Care Medicine, Railway Hospital and Clinic Košice, Slovak Republic 6 EuroPainClinics, Košice, Slovak Republic 7 Pain Clinic, Faculty Hospital A. D. Roosevelt Banská Bystrica, Slovak Republic 8 Department of Medical and Clinical Biochemistry, Faculty of Medicine, Pavol Jozef Šafárik University, Košice, Slovak Republic ft rA Chronic limb-threatening ischemia (CLTI) is the end stage of lower extremity artery disease with rising prevalence [1]. The prognosis of CLTI patients is alarming and even more K devastating when revascularization is not possible [2]. One of the supportive treatment possibilities for CLTI patients is lumbar sympathectomy (LS) causing irreversible damage to a part of the lumbar sympathetic trunk, thus S decreasing peripheral resistance of lower limb arteries con- trolled by sympathetic activity [3]. The LS is standardly peril formed on the desired side according to vascular pathology at the L3 spinal level. However, it causes anatomically non-£> specific blockade of the lumbar sympathicus, thus it can m often block both sides. 2 The angiosome is defined as a three-dimensional network of vessels in all tissue layers between the skin and bone. Two types of anastomotic arteries between adjacent §. angiosomes were identified: similar-caliber ("true") anas- - tomotic arteries and reduced-caliber ("choke") that ordi- narily exist in a state of reduced caliber during routine circulation and are controlled by sympathetic tone [4]. In the most common angiosome model, the foot consists of six angiosomes originating from the 3 main source arteries: posterior tibial artery (PTA), anterior tibial artery (ATA) and peroneal artery (PA). Dermatomes represent the surface area of angiosomes. Therefore it makes sense to evaluate perfusion in angiosomes by temperature changes in dermatomes caused by vasodilatation enhanced due to sympathectomy. Anatomical relationship between dermatomes and angiosomes of the foot shows Figure 1. DPA+MPAHPA Figure 1. Anatomical relationship between angiosomes and dermatomes of the foot. (A) Angiosomes. MPA: angiosome of the medial plantar artery; LPA: angiosome of the lateral plantar artery (MPA and LPA are perfused by the posterior tibial artery): CBPTA: calcaneal branch of posterior tibial artery (PTA) angiosome (MPA, LPA and CBPTA create together one angiosome perfused by PTA): DPA: angiosome of the dorsalis pedis artery (angiosome perfused by the anterior tibial artery); PA: peroneal artery angiosome. (B) Dermatomes. SC: lateral sural cuta-neous nerve (L5-S2); CC: saphenous medial crural cutaneous nerve (L3-4); SP: superficial peroneal nerve (L4-S1); DP: deep peroneal nerve (L4-5); LP: dermatome of the lateral plantar nerve (S1-2); MP: dermatome of the medial plantar nerve (L4-5); T: dermatome of the tibial nerve (SI -2); S: dermatome of sural nerve (S1 -2). SP and DP create together the surface area of DPA/ATA angiosome. LP, MP and T create together the surface area of the PTA angiosome. S is the surface area of the PA angiosome. We have hypothesized, that alteration in sympathetic tone could cause dilation and reorientation of flow in choke vessels and this way sympathectomy may increase the blood flow from adjacent angiosomes with patent arteries to an angiosome perfused by an occluded arteries which should be associated with an increase of temperature [5]. 3 2023 Hogrefe Voso (2023). 52 (2), 133-135 https://doi.org/! 0.1024/0301 -1526/a001053 109 Letter to the editor Before procedure 10 minutes after procedure 20 minutes after procedure Figure 2. Images of thermal changes of the foot in Patient A, B and C taken by FLIR SC660 thermal image camera before. 10 minutes and 20 minutes after the procedure with detailed area segmentation. R: right side of the patient; L: left side of the patient. 123 123 123 123 1 1 - Before procedure 2 = 10 minutes after procedure 3 = 20 minutes after procedure Figure 3. Line graphs showing changes of temperatures in each foot dermatome of all patients before, 10 minutes and 20 minutes after the procedure (LS). Vosa (2023). 52 (2). 133-135 ©2023 Hogrefe 110 Letter to the editor 135 < 1 In our pilot study, we measured changes in foot temperature by Infrared thermography before LS, than 10 and 20 minutes after LS in three CLTI patients (A, B, C) with chronic lower limb wound and ischemia at least grade 1 according to WIfl classification. Clinically relevant change in temperature was arbitrarily considered an increase of more than 1 °C after the procedure. The study protocol was approved by an ethical committee. All patients underwent angiography before LS. Patient A had three vessels runoff on the right limb and 2 vessels runoff - ATA and PA, PTA was occluded on the left limb. Angiographic findings of patient B were similar on both limbs, PTA was bilaterally occluded. Patient C had domi-nantly venous wounds due to post thrombotic syndrome. The severity lesions of below-the-knee arteries were up to 50% bilaterally in this patient. Comparing the left and right foot, a relevant increase in temperature was observed 20 minutes after the procedure in patient A on the left foot in lateral and medial plantar dermatomes ranging from 1.7 °C to 2.1 °C (Figures 2 and 3) that corresponds to PTA angiosome. In patient B, there was an apparent change in temperature from 1.5 °C to 2.1 °C (left foot) and from 1.2 °C to 3.0 °C (right foot) in lateral, medial plantar, and tibial areas bilaterally (Figures 2 and 3) that also corresponds to PTA angiosome. Patient C had no relevant increase in temperature (Figures 2 and 3). Improvement in wound healing and partial reduction of wound size was observed in patients A and B only. We have documented a relevant increase of temperature after LS only in dermatomes which create surface areas of angiosomes perfused by occluded arteries, probably due to the opening of choke vessels after the LS procedure. However, our findings are too preliminary and need to be verified by another study with a higher sample size. Despite this, we have performed LS in several CLTI patients with no option for revascularization (but already without thermal imaging). References 1. de Graaff JC. Ubbink DT, Legemate DA, de Haan RJ, Jacobs MJ. hterobserver and intraobserver reproducibility of peripheral blood and oxygen pressure measurements in the assessment of lower extremity arterial disease. J Vase Surg. 2001 ;33(5): 1033-40. 2. Wang Z. Hasan R, Firwana B, Elraiyah T, Tsapas A, Prokop L, et al. A systematic review and meta-analysis of tests to predict wound healing in diabetic foot. J Vase Surg. 2016:63(2 Suppl): 29S-36S.e1-2. 3. Cronenwett JL. Lindenauer SM. Direct measurement of arteriovenous anastomotic blood flow aMer sympathectomy. Surgery. 1977:82(1):S2-9. 4. Taylor Gl, Palmer JH. The vascular territories (angiosomes) of the body: experimental study and clinical applications. Br J Plast Surg. 1987;40(2):113-41. 5. Taylor Gl, Palmer JH. Angiosome theory. Br J Plast Surg. 1992:45:327-8. History Submitted: 07.11.2022 Accepted after revision: 10.01.2023 Published online: 06.03.2023 Conflict of Interest There are no conflicts of interest existing. ORCID Viktoria Rajťúková © https://orcid.org/0000-0002-7180-2874 Radovan Hudák © https://orcid.org/0000-0003-1060-0539 Dušan Rybár © https://orcid.org/0000-0003-0803-3213 Janka Vašková ©https://orcid.org/0000-0003-0465-7950 Marek Hudák © https://orcid.org/0000-0003-0820-1328 Correspondence address Marek Hudák, MD, PhD East Slovak Institute of Cardiovascular Diseases: Východoslovensky Ustav Srdcových a Cievnych Chorob AS Ondavská 8 04011 Košice Slovakia mhudak@vusch.sk © 2023 Hogrefe Vaša (2023), 52 (2). 133-135 111 Ďalšie práce spojené s ambulantnou algeziologickou prácou boli publikované v recenzovaných medicínskych časopisoch Radiology Case Reports Q3, Neurologie pro praxi, Polish Journal of Sports Medicine Q4 a časopise Bolest: • Kočan L, Rapčan R, Griger M, Rapčanová S, Kantárová D, Tôrôk P, Vašková J. Deciphering the enigmatic symptoms of Pancoast tumors: Navigating the complex landscape of pain management-A case report. Radiol Case Rep. 2024Feb 20;19(5):1810-1814. doi: 10.1016/j.radcr.2024.01.091. • Dvorak M, Zelinský Ľ, Horný V, Rapčan R, Neuwirth J, Griger M, Kočan L. Multidisciplinárny prístup v diagnostike vertebrogénnych ochorení z pohľadu neurológa a fýziatricko-rehabilitačného lekára. Neurol, praxi. 2019;20(6) :417-420. ISSN 1213-1814. • Neuwirth J, Griger M, Rapčan R, Dvorak M, Zelinský Ľ, Horný V, Kočan L. Multidisciplinárny prístup v diagnostike vertebrogénnych ochorení z pohľadu radiologa a intervenčného algeziológa. Neurol, praxi. 2019;20(6):423-427. ISSN 1213-1814. • Mláka J, MatiasM, Rapčan R, Rapčanová S, Kočan L. Perkutánne endoskopické techniky v diagnostike a liečbe vertebrogénnych ochorení: epiduroskopia a endoskopická diskektómia. Neurol, praxi. 2019;20(6):428-432. ISSN 1213-1814. • Rapčan R, Martuliak I, Lejčko J, Illéš R, Krajčovič M, Kočan L. Neuromodulačné techniky v riešení vertebrogénnych bolestivých syndrómov. Neurol, praxi. 2019;20(6):433-438. • Rapčan R, Poliak Ľ, Rapčanová S, Lenčéš P, Burianek M. Vašková J. Kočan L. The influence of various sport activities on the degeneration of intervertebral discs. Polish Journal of Sports Medicine. 2023. 3. 115-121. ISSN 1232-406X. • TirpákR, Rapčan R, Griger M, Mláka J, Burianek M, Poliak Ľ, LenčešP, MatiasM, Kočan L. Terapeutické možnosti a technika periradikulární terapie při kořenových syndromech. Bolest. 22. č 1. 2019. 21-28. ISSN 1212-0634. 112 • Poliak Ľ, Rapčan R, Griger M, Mláka J, Burianek M, Tirpák R, Lenčeš P, Matias M, Kočan L. Komplexná liečba radikulárneho syndrómu (prehľad terapeutických možností). Bolest. 22 č.2. 2019. 1-8. ISSN 1212-0634. Výsledky intervenčných techník zameraných na liečbu chronickej bolesti chrbta u pacientov s pozitívnym fazetovým syndrómom sme sledovali v multicentrickej randomizovanej klinickej štúdii EPCS Xlr NCT04684303. V štúdii sme skúmali efektivitu intervenčných výkonov u pacientov s bolesťou dolnej časti chrbta po pozitívnom testovaní zameranom na mediálně nervové ramienka dorzálnej vetvy spinálneho nervu, ktoré inervujú fazetové kíby v lumbosakrálnej oblasti. Po diagnostikovaní lumbálneho fazetového syndrómu, splnení inklúznych kritérií pre klinickú štúdiu boli pacienti randomizáciou rozdelení do 3 skupín podľa nasledujúceho flow diagramu. Low back pain RTG testovacia blokáda Pozitivny Iumba lny fazetový syndrom random izácia Kryoablácia medtálneho nervováno ramienka spinálneho nervu Rádiofrekvenčná ablácia mediálneho nervového ramienka spinálneho nervu Parciálna endoskopicka dekapsulácia fazetového kíbu Obrázok č.10: Flow diagram klinickej štúdie EPCS Xlr. Výsledky prezentované na kongrese World Institute of Pain - WTP XI WORLD CONGRESS BUDAPEST 2022, poukázali na signifikantné zlepšenie parametrov (p < 0.05) bolesti dolnej časti chrbta, ako aj bolestí vyžarujúcich do dolných končatín a signifikantného zlepšenia hodnotených dotazníkov kvality života Oswestry a EuroQuol v časových obdobiach po 3, 6, 113 a 12 mesiacoch od realizovaného výkonu. Zároveň medzi skupinové porovnanie medzi skupinami kryoablácia (n =70), rádiofrekvenčná ablácia (n=40) a endoskopická parciálna dekapsulácia (n=50) nepotvrdili signifikantně rozdiely vo všetkých sledovaných časových periódach p > 0.05. Porovnanie metodických postupov rádiofrekvenčnej ablácie akryoablácií kolmým a paralelným uložením kryosondy bolo publikované v medicínskom časopise Radiology Case Reports IF 1,0, Q4. • Kočan L, Rapčan R, Sudzina R, Rapčanová S, Rybár D, Mláka J, Kočanová H, Buriánek M, Vašková J. Radiofrequency denervation and cryoablation of the lumbar zygapophysial joints in the treatment of positive lumbar facet joint syndrome - a report of three cases. Radiol Case Rep. 2022 Sep 26;17(12):4515-4520. doi: 10.1016/j.radcr.2022.09.010. 114 Radiology Case Reports 17 (2022) 4515-4520 RCR RADIOLOGY CASE REPORTS ELSEVIER Available online at www.sciencedirect.com ScienceDirect journal homepage: www.elsevier.com/locate/radcr Case Report Radiofrequency denervation and cryoablation of the lumbar zygapophysial joints in the treatment of positive lumbar facet joint syndrome - a report of three cases Ladislav Kočan, MD, PhDa'b, Robert Rapčan, MD, PhDc, Rudolf Sudzina, MD, PhDd, Simona Rapčanová, MDC, Dušan Rybár, MDa, Juraj Mláka, MDb, Hana Kočanová, MDe, Miroslav Buriánek, MEŕ, Janka Vaškouá, PhD9'* 3 Clinic of Anesthesiology and Intensiue Care Medicine, East Slouafe institute of Cardiovascular Disease, Ondavská 8, 040 11 Košice, Slouak Republic bEuroPainCiinics, Poliklinika Terasa, Toryská 1, 040 11 Košice, Slouak Republic cEuroPainCIinics, Na hradbách 94/3, 085 01 Bardejou, Slouak Republic d Neurological Clinic, East Slouak Institute of Cardiovascular Disease, Ondauská 8, 040 11 Košice, Slouak Republic e Clinic of Anesthesiology and Intensiue Care Medicine, Railiuay Hospital and Clinic Košice, Slouak Republic fEuroPainClinics, Starochodouská 1750/91,149 00 Prague 4, Czech Republic s Department of Medical and Clinical Biochemistry, Faculty of Medicine, Pauol Jozef 3a_fárik Uniuersity in Košice, TV. SNP 1, 040 11, Košice, Slouak Republic ARTICLE INFO ABSTRACT Article history: Received 27 July 2022 Revised 30 August 2022 Accepted 4 September 2022 Available online 26 September 2022 Keywords: Cryoablation Facet joint Radiofrequency ablation Zygapophysial joint Radiofrequency denervation of the zygapophysial (facet) joints is a frequently performed procedure for chronic low back pain. However, cryoablation represents a novel therapeutic approach for this condition. We observed and analyzed 3 cases with confirmed positive lumbar facet joint syndrome. Our results show a significant improvement in the clinical state of the patients in the first and third months after the procedure. The 6-month follow-up examination demonstrates a recurrence of pain and a gradual deterioration in the quality of life with a lasting partial pain-relief effect. Thermal radiofrequency denervation and cryoablation of the lumbar zygapophysial joints represent an effective, albeit temporary treatment option for lumbar facet joint syndrome patients, resulting from the pathophysiology of sensory nerve regeneration after destructive procedures. This type of treatment can be used repeatedly in the case of a positive response. © 2022 The Authors. Published by Elsevier Inc. on behalf of University of Washington. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4-0/) !* Competing Interests: None of the authors has financial/commercial conflicts of interest with the published data. Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. * Corresponding author. E-mail address: janka.vaskova@upjs.sk (J. Vašková). https://doi.Org/10.1016/j.radcr.2022.09.010 1930-0433/© 2022 The Authors. Published by Elsevier Inc. on behalf of University of Washington. This is an open access article under the CC BY-NC-ND license {http://creativecornmons.Org/licenses/by-nc-nd/4.0/) 115 4516 Radiology Case Reports 17 (2022) 4515-4520 Introduction Back pain is the most common type of pain in humans. More than 80% of the global population will experience low back pain at least once in their life and it is considered chronic if it lasts 3 months or longer [1-3]. The main cause is degenerative changes of spine structures including the facet joints, which can be a potentially important source of symptoms because of the high level of mobility and load forces, especially in the lumbar area [4,5]. Treatment of facet joint syndrome is mul-tidisciplinary, and when conservative methods are not sufficient, radiofrequency thermal ablation is performed. More recently, cryoablation has emerged as an alternative interventional treatment option of facet joint syndrome [6,7], In the following 3 case studies, we describe the treatment of facet joint syndrome using radiofrequency thermal ablation and cryoablation techniques placing the electrodes perpendicularly and in parallel with the nerve course. Cryoablation was performed with a CRYO-S PAINLESS device with carbon dioxide (-78°C) as a working medium for 2 minutes in 2 cycles for one nerve. This device also contains an integrated neu-rostimulator. When proper cryoprobe position was confirmed in multiple views, the impedance was checked and followed by sensory stimulation with a current of 50 Hz and motor stimulation with a current of 2 Hz. We followed these patients before the procedure, 4 weeks, 12 weeks, and 6 months after the procedure. Measured parameters included lower back pain intensity and intensity of pain of the lower extremities based on the Numeric Rating Scale (NRS) from 0 to 10. We used quality of life questionnaires, Oswestry Disability Index (ODI), which is focused on performing everyday activities affected by lower back pain, where the score 0 means the best possible status and 100 the worst possible status. We also used the licensed EQ-5D-5L questionnaire, evaluating the quality of life through 5 dimensions: mobility, self-care, everyday activities, pain/discomfort, and anxiety/depression. Each dimension is rated by the patient from 1 to 5, where a score of 1 means the best condition and a score of 5 means the worst possible con- dition. The EQ-VAS (EQ visual analogue scale) describes the current health condition, where 0 means the worst possible state, and 100 means the best possible state. This clinical trial was approved by the ethical committee of the Medical Faculty of the Pavol Jozef Safarik University in Kosice, with the registration number 75/EK/15, as well as being registered in the international database clinicaltrials.gov under the registration number NCT03039296. All patients signed an informed consent about the participation in the study and publishing the clinical results. Case series Patient 1 Seventy-one-year-old patient, pensioner, with chronic lower back pain in the lumbo-sacral area after 6 spinal surgeries in the past, currently on combined pharmacological therapy: buprenorphine transdermal patch 35 ug/h and pregabalin 75 mg twice daily. In the past, the patient had repeated caudal pressure blocks (5 in total) with minimal therapeutic effect. The first MRI (magnetic resonance imaging) examination describes a multilevel herniation of the intervertebral discs L3/L4, L4/L5, listhesis of the L5 vertebra, and multilevel bilateral facet joint arthritis. Currently, the patient is experiencing a dull pain in the lumbo-sacral area with an NRS intensity of 9/10 radiating to the lower extremities. The patient underwent bilateral test blockade of the medial branches of the dorsal ramus of the spinal nerve (DRSN) at the interventional pain management clinic, after which they described 100% pain relief lasting for 48 hours. Subsequently, the patient underwent bilateral radiofrequency denervation of the medial branches of the DRSN innervating the facet joints L3/L4, L4/L5, and L5/S1. The radiofrequency probe was placed in parallel with the anticipated anatomical course of the sensory nerve (Fig-1). Fig. 1 - Final placement of the radiofrequency electrodes at the L3/L4 and L4/L5 facet joint level left side, anterior and oblique projections. Radiology Case Reports 17 (2022) 4515-4520 4517 Patient 2 Forty-five-year-old patient working as a teacher and artistic carpenter was repeatedly hospitalized at the neurological ward for attacks of back pain in the lumbosacral area. The current MRI of the low back spine shows multilevel discopa-thy, moderate ventrolisthesis of L5t minimal dorsolisthesis of L3 to L4. Further, it shows an L5/S1 intervertebral disc herniation affecting the posterior lower border of the L5 vertebra with the size 5.8 mm and a multilevel herniation. The patient has intense pain of their lumbar spine area radiating to the right lower limb, paresthesias on the lateral border of their thigh, they have problems being seated for a longer period of time. The patient has been using NSAIDs (nonsteroidal anti-inflammatory drugs) in combination with Tra-madol/Paracetamol tablets during intense bouts of pain. After a bilateral test blockade, the patient felt 70% relief of pain for 12 hours. They then underwent cryoablation of the medial branches of the facet joints L3/L4, L4/L5, L5/S1. The cryoprobe was placed perpendicularly to the expected anatomical course of the sensory nerve (Fig. 2). Patient 3 Sixty-four-year-old patient with a desk job, complains of intense pain of their lumbar spine radiating to both lower limbs, more on the right, lasting approximately 4 years. The pain occurs mainly at night. The current MRI shows no compression of any structures, numerous Schmorl's nodes in vertebral bodies, L3/L4 and L4/L5 segments shows MODIC1 and MODIC2 (modic type endplate changes, status 1 and 2) changes. Clinical examination revealed pseudoradicular syndrome, provocation tests for the SI joins were negative without a sensory or motor deficit in the lower limbs. Analgesic therapy consisted of sporadic use of NSAIDs and metamizol. The patient underwent a bilateral test facet joint blockade, with significant pain relief about 90% directly after the procedure lasting up to the night hours. Here, we performed a bilateral thermal ablation of the medical branches (DRSN) L3, L4, and L5 with cryoanal-gesia. The cryoprobe was placed in parallel with the expected anatomical course of the sensory nerve (Fig. 3). In all cases, we noted a significant improvement of pain intensity in the 1st and 3rd months after the procedure with a lasting pain relief effect or slightly recurring pain at the 6 months follow-up (Table 1). Similar results were noted in patients monitored by ODI, EQ-VAS, and the EQ-5D-5 dimensions with a lasting positive treatment effect as well as a slight worsening at the 6 months follow-up (Tables 1 and 2). Discussion Facet joint syndrome is linked with the dysfunction in the joint of the same name, which becomes the source of pain. The most common causes of this are trauma, more commonly repeated microtrauma in the facet joint area caused by repeated extension of the lumbar spine or with overhead activities, as well as sports involving tiresome extension exercises of the lumbar spine. One of the most common causes is the damage of the intervertebral disc, which causes a disruption in the biomechanics of the facet joints. This can cause their subluxation, microtrauma, swelling of the synovium surrounding the facet joints, leading to synovitis. A hypertonic contraction of surrounding structures occurs as a defence mechanism, which leads to the worsening of pain. An important step in the diagnosis of lumbar facet joint syndrome is a test blockade of the medial branch DRSN. Precise diagnostics is important, similarly as it was performed in patients from the case series where, after injecting a small amount of local anesthetic (0.5 mL 0.5 % bupivacaine) under X-ray guidance to the sensory medial branch DRSN, significant pain relief was achieved for numerous hours. This test was performed twice in each patient. After a positive test result, cryoablation or radiofrequency denervation was performed, which provided 4518 Radiology Case Reports 17 (2022) 4515-4520 Fig. 3 - The final parallel placement of the cryoprobe to the L4/L5 facet joint on the left in the AP projection and parallel placement of the cryoprobe to the L4/L5 facet joint on the right, shown in the oblique projection. Table 1 - Trend of change in the clinical status in the Oswestry Disability Index (ODI) and EQ-VAS (EQ visual analogue scale) parameters and pain intensity assessed by the Numerical Rating Scale (NRS) at each time interval in the lumbo-sacral back pain and pain radiating to the lower extremities followed in patients 1, 2, and 3. Patient (numberj/technical aspects Parameters Follow-up Before procedure 1 month 3 month 6 month Radiofrequency ablation ODI 45 5 9 22 EQ-VAS 50 90 80 60 NRS back pain 9 2 1 5 NRS leg pain 3 1 1 2 Cryoablation perpendicularly ODI 63 31 31 45 EQ-VAS 75 85 85 55 NRS back pain 8 4 4 6 NRS leg pain 8 5 5 8 Cryoablation in parallel ODI 56 4 9 29 EQ-VAS 55 70 85 65 NRS back pain 7 4 2 7 NRS leg pain 7 4 4 6 the patient with pain relief for a couple of months. Both of these procedures cause targeted physical damage of the nerve in a limited area, either by the action of an electromagnetic field, increased temperatures in the desired area, or cold temperatures. These physical effects cause reversible damage to the nerve, a so-called axonotmesis, where axonal damage occurs with the preservation of the epineurium. During this damage, a defect in the transmission of nerve signals occurs, which clinically manifests as the disappearance of pain. Dis-tally from the nerve damage, Wallerian degeneration occurs, as well as changes of the myelin sheath, but the periphery of the nerve maintains its electrical irritability for 5-10 days. After a couple of days, the body of the neuron is activated -sprouting; each nerve fiber from the damaged axon regenerates at the speed of 1 mm per day [7,8]. The return of pain after neural ablation indicates nerve regeneration after peripheral nerve damage after a couple of weeks or months. This trend of pain recurrence was demonstrated in all patients. In clinical practice, we are also met with unsuccessful thermal ablation procedures. This can have numerous causes. The most common cause is multifactorial etiology of lower back pain. Facet joint arthropathy is often connected with discopa-thy, radiculopathy, spinal canal stenosis, and myofascial syndrome. The co-existence of these co-morbidities means that treating only one of them can lead to unsuccessful pain relief. Another cause can be a false positive diagnostic blockade, by injecting too much local anesthesia and its spread to the surrounding structures, which could also be the sources of pain [9-11]. A high percentage of positive reactions after a placebo dose are the cause of false positive results [12]. Aberrant nerve sprouting could also be the cause of unsuccessful treatment. Okuyama et al. discovered, that after RF ablation of the myocardium, aberrant nerve sprouting occurs already after 2 hours, and it is probable that the same process occurs after RF ablation of the medial branches of the DRSN [13,14]. The choice of facet joint thermal ablation technique (radiofre- Radiology Case Reports 17 (2022) 4515-4520 4519 Table 2 - Trend of changes in the patients' assessment of everyday activities at each time interval, where a score of 5 indicates the worst possible status and the score 1 means the best possible status. Patient/technical aspect Mobility/ follow-up Before procedure 1 month 3 month 6 month Radiofrequency ablation 4 2 2 3 Cryoablation perpendicularly 4 2 2 3 Cryoablation in parallel 3 Self-care/ follow-up 1 2 2 Radiofrequency ablation 3 1 1 3 Cryoablation perpendicularly 3 2 2 3 Cryoablation in parallel 4 Usual activities/follow-up 1 1 2 Radiofrequency ablation 4 2 2 3 Cryoablation perpendicularly 4 2 1 3 Cryoablation in parallel 4 Pain and discomfort/follow-up 2 2 2 Radiofrequency ablation 4 1 2 3 Cryoablation perpendicularly 3 1 2 3 Cryoablation in parallel 4 1 2 2 Anxiety and depression/follow-up Radiofrequency ablation 3 1 1 2 Cryoablation perpendicularly 3 1 2 3 Cryoablation in parallel 4 1 2 3 quency or cryoablation) as well as probe placement can also have an influence on the final treatment effect, seeing as they have different nerve damage mechanisms. The choice of interventional thermal ablation method is not strictly defined, seeing as there have been no published EBM results on the superiority of one technique over the other. In terms of the result, the shape and extent of the lesion which forms around the tip of the electrode are important, and they depend on its physical properties and treatment procedure. Placement of the RF electrode during radiofrequency denervation is defined by SIS guidelines based on EBM, which consists of placing the probe in parallel with the anatomic course of the nerve [1]. The reason is to damage the nerve the most efficiently. Placement of the cryoablation electrode is not yet clearly determined, which is why in our patients, we placed them in the 2 most common used locations in practice - perpendicular to and in parallel with the nerve course. Conclusion Due to the diverse etiology of lower back pain, it is imperative to determine the cause of the pain to plan the treatment procedure. To correctly determine the source of lower back pain, we have to perform a complex evaluation. In a precisely diagnosed lumbar facet joint syndrome, thermal ablation techniques represent a relevant therapeutic tool. Patient consent Written informed consent for the publication of this case report was obtained from each patient. REFERENCES [1] Manchikanti L, Pampati V, Fellows B, Bakhit CE. Prevalence of lumbar facet joint pain in chronic low back pain. Pain Phys 1999;2(3):59-64. [2] Bogduk N. Practice guidelines for spinal diagnostic and treatment procedures. San Francisco: International Spine Intervention Society, United States; 2013. p. 684. (in Eng.). [3] Geraci MC, Baker RM, Sasso RC. Lumbar zygapophysial joint evaluation and treatment. Curr Concepts 2005:10-17. [4] Lau P, Mercer S, Govind J, Bogduk N. The surgical anatomy of lumbar medial branch neurotomy (facet denervation). Pain Med 2004;5(3):289-98. doi:10.1111/j.l526-4637.2004.04042.x. 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Clin } Pain 1995;11(3):208-13. doi:10.1097/00002508-199509000-00008. [13] Okuyama Y, Pak HN, Miyauchi Y, Liu YB, Chou CC, Hayashi H, et al. Nerve sprouting induced by radiofrequency catheter ablation in dogs. Heart Rhythm 2004;1(6):712-17. doi:10.1016/j.hrthm.2004.09.012. [14] Kim MH, Kim SW, Ju CI, Chae KH, Kim DM. Effectiveness of repeated radiofrequency neurotomy for facet joint syndrome after microscopic discectomy. Korean J Spine 2014;ll(4):232-4. doi:10.14245/kjs.2014.11.4.232. 6.3 Multicentrické projekty spojené s liečbou a výskumom akútnej a chronickej bolesti Ďalšou dôležitou činnosťou zameranou na liečbu bolesti je aktívna spolupráca s viacerými algeziologickými ambulanciami, klinikami neurológie a neurochirurgie v rámci Slovenska a Českej republiky. Významná spolupráca prebieha s Ústavom merania Slovenskej Akadémie Vied (UM SAV). Ako dôležitý výskumný počin považujeme vytvorenie a vedenie multicentrických klinických štúdií zameraných na hodnotenie kvality života u pacientov s akútnou a chronickou bolesťou po intervenčných algeziologických výkonoch, a to prostredníctvom dotazníkov Oswestry Disability Index a EuroQuol EQ-5D-5L. V súčinnosti s UM SAV sme stanovili koeficienty štatistických váh pre štatistickú analýzu dotazníkov EQ 5D-5L na modeloch skupín pacientov s akútnou a chronickou bolesťou pre regióny Česka a Slovenska. Túto metodiku je možné ďalej využívať pre hodnotenie kvality života rôznych diagnóz v rozličných medicínskych odboroch. Medzi naše hlavné klinické štúdie, v ktorých bola hodnotená kvalita života pacientov patria: Prospektívna randomizovaná multicentrická klinická štúdia EPCS II (zameraná na chronickú bolesť u pacientov s postlaminektomickým syndrómom - „failed back surgery syndrome" - FBSS) a Prospektívna observačná klinická štúdia EPCS V (zameraná na liečbu akútnej bolesti u pacientov s akútnym radikulárnym syndrómom, spôsobeným útlakom koreňa herniovaným diskom alebo sequestrom). Výsledky štúdie EPCS II boli publikované v medicínskych časopisoch: Pain Medicíne IF 3,1, Ql, Wiener klinische Wochenschrift IF 2,6 Q2 a Bratislava Medical Journal, IF 1,4, Q4. Výsledky poukazujú na klinický benefit u pacientov s FBSS po epiduroskopickom výkone, a to v zlepšení intenzity bolesti v dolnej časti chrbta a výsledkov dotazníkov Oswestry a EuroQuol po 6 mesiacoch (p < 0.05). V skupine, v ktorej bola vykonaná epiduroskopia a simultánna aplikácia liečiv do epidurálneho priestoru sme zaznamenali signifikantně predĺžený pozitívny efekt v parametri intenzita bolesti vyžarujúcej do dolných končatín po 12 mesiacoch (p < 0.05). 121 Najdôležitejšie publikácie štúdie EPCS II: • Griger M, Kocan L, Rapcan R, Matias M, BurianekM, Kocanova H, Rapcanova S, Mláka J, Zahorec R, VaskovaJ. Epiduroscopic intervention in patients with a failed back surgery syndrome. BratislLekListy. 2020; 121(10):727-732. doi: 10.4149/BLL 2020 119. IF 1,7, Q3 • Rapčan R, Kočan L, Witkovsky V, Mláka J, Griger M, Burianek M, Rapčanová S, Hammond A, Poliak Ľ, Tirpák R, Simonová J, Sabol F, Vašková J. EQ-5D-5L questionnaire as suitable assessment of quality of life after epiduroscopy : Multicenter randomized double-blind pilot study. Wien Klin Wochenschr. 2020 Sep;132(17-18):526-534. doi: 10.1007/s00508-019-01590-z. IF 1,6, Q3 • Rapčan R, Kočan L, Mláka J, BurianekM, Kočanová H, Rapčanová S, Hess M, Hammond A, Griger M, Venglarčík M, Gajdoš M, Vašková J. A Randomized, Multicenter, Double-Blind, Parallel Pilot Study Assessing the Effect of Mechanical Adhesiolysis vs Adhesiolysis with Corticosteroid and Hyaluronidase Administration into the Epidural Space During Epiduroscopy. PainMed. 2018Jul 1;19(7):1436-1444. doi: 10.1093/pm/pnx328. IF2,8 Ql Výsledky štúdie EPCS II boli citované v Metaanalýzach, Systémových prehľadoch, a medzinárodných odporúčaniach: • Geudeke MW, Krediet AC, Bilecen S, Huygen FJPM, Rijsdijk M. Effectiveness of Epiduroscopy for Patients with Failed Back Surgery Syndrome: A Systematic Review and Meta-analysis. PainPract. 2021 Apr;21(4):468-481. doi: 10.1111/papr. 12974. • Papalia GF, Russo F, et al. V. Non-Invasive Treatments for Failed Back Surgery Syndrome: A Systematic Review. Global Spine J. 2023 May;13(4):1153-1162. doi: 10.1177/21925682221141385. • Manchikanti L, Racz GB, , et all. Epidural Interventions in the Management of Chronic Spinal Pain: American Society of Interventional Pain Physicians (ASIPP) Comprehensive Evidence-Based Guidelines. Pain Physician. 2021 Jan;24(Sl):S27-S208. PMID: 33492918. 122 Pain Medicine 2018; 19: 1436-1444 doi: 10.1093/pm/pnx328 oxford NEUROMODULATION & INTERVENTION SECTION Original Research Article A Randomized, Multicenter, Double-Blind, Parallel Pilot Study Assessing the Effect of Mechanical Adhesiolysis vs Adhesiolysis with Corticosteroid and Hyaluronidase Administration into the Epidural Space During Epiduroscopy Róbert Rapcan, MD,*,Ť'* Ladislav Kočan, MD, PhD,§ Juraj Mláka, MD,11 Miroslav Burianek, MD, MBA,* Hana Kocanova. MD,1 Simona Rapcanova, MD,* Michael Hess, MD, Anthony Hammond, MD,** Martin Griger, MD,+ Michal Venglarčík, M D, PhD,* Miroslav Gajdoš, MD, PhD,Ťt and Janka Vašková, PhD** "Europainclinics, Prague, Czech Republic; tEuropainclinics, Nové Mesto, Slovak Republic; *Europainclinics, Bardejov, Slovak Republic; §Clinic of Anaesthesiology and Intensive Care Medicine, East Slovak Institute of Cardiovascular Disease, Košice, Slovak Republic; 'Europainclinics, Poliklinika Terasa, Košice, Slovak Republic; "Clinic of Anaesthesiology and Intensive Care Medicine, Railway Hospital and Clinic Košice, Košice, Slovak Republic; 11 Wirbelsäuleninstitut, Munich, Munich, Germany; "Kent Institute of Medicine and Surgery, Kent, UK; 'ŤDepartment of Neurosurgery, Faculty of Medicine, Pavol Jozef Šafárik University in Košice, and Louis Pasteur University Hospital, Košice, Slovak Republic; '^Department of Medical and Clinical Biochemistry, Faculty of Medicine, Pavol Jozef Šafárik University in Košice, Košice, Slovak Republic Correspondence to: Janka Vašková, PhD, Department of Medical and Clinical Biochemistry, Faculty of Medicine, Pavol Jozef Šafárik University in Košice, Trieda SNP 1, 040 66 Košice, Slovak Republic. Tel: 421552343232; Fax: 421552343401; E-mail: janka.vaskova@upjs.sk. Conflicts of interest: None of the authors has any financial/commercial conflicts of interest with the published data. Abstract Objective. Epiduroscopy is a proven method of diagnosis and treatment for chronic radicular pain after spinal surgery, which is known as failed back surgery syndrome (FBSS). The aim of the study was to compare the efficacy of drugs (the enzyme hyaluronidase and corticosteroid DEPO-Medrol) administrated into the epidural space during epiduroscopy, performed within the ventral and ventro-lateral epidural space with a focus on releasing foraminal adhesions. Methods. Forty-eight patients with diagnosed FBBS were randomized into two groups before epiduroscopy. Group A received the standard treatment—mechanical lysis of fibrotic tissue in the epidural space. Group B received hyaluronidase and corticosteroid methylprednisolone acetate during the procedure. Subjects were followed for six and 12 months via scheduled double-blinded examinations by pain physicians. Leg and back pain intensity was assessed by an 11-point numerical rating scale, and patients' functional disability was assessed by the Oswestry Disability Index (ODI). Results. Study subjects showed a significant decrease in ODI score in both groups (P<0.05). Significantly lower pain scores for leg pain (P<0.05) and back pain (P<0.05) were also recorded after the six-month follow-up. However, the one-year follow-up showed a return to the baseline ODI values of most monitored pain scores in both groups (P>0.05). Improvement was only noted on the NRS for back pain at one-year follow-up (P< 0.05). Conclusions. A significant improvement of leg and back pain was found in both groups after six 2018 American Academy of Pain Medicine. Al! rights reserved. For permissions, please e-mail: journals.permissions@oup.com 1436 Drug Administration During Epiduroscopy months. ODI was significantly improved only in group B in both the six- and 12-month intervals. Back pain at one-year follow-up was only improved in group B. Key Words. Adhesiolysis; Back Pain; Epiduroscopy; Failed Back Surgery Syndrome; Hyaluronidase Introduction Medical experts and researchers have investigated various types of optical visualization of human cavities for decades, with varying degrees of success [1]. Epiduroscopy is a relatively new technique used in the evaluation and treatment of low back pain via advancements in optical fiber technology. As a minimally invasive, endoscopic technique, it allows for direct endoscopic imaging of the epidural space and helps with pain management for patients suffering from post-lumbar surgery syndrome (PLSS) and other causes of low back pain and radiculopathy [2]. Epiduroscopy, the direct visualization of the epidural space with a flexible endoscope, has been performed in some places for years, but its significance is still questionable. For example, it has been shown to be more sensitive than magnetic resonance imaging (MRI) in detecting epidural fibrosis. According to literature, in patients with failed back surgery syndrome, MRI showed epidural fibrosis in 16.1% of patients whereas epiduroscopy showed epidural fibrosis in 91 % of patients [3]. A systematic review of the literature regarding the effectiveness of spinal endoscopic adhesiolysis in managing chronic intractable pain from PLSS indicated an evidence level of 11-1 or II-2 based on the US Preventive Services Task Force (USPSTF) criteria, and one randomized trial gave it a recommendation of 1 C/strong [4]. The authors of this study have been using epiduroscopy for a number of years, and they see a potential for this method to enable physicians to perform a detailed examination of the spinal canal with optic visualization of the particular structures. This type of examination will provide detailed information about the presence of postoperative fibrosis, adhesions, inflammatory changes, or any other pathological change within the epidural space. The current technique also allows the utilization of optical visualization for targeted therapeutical interventions in the epidural space, such as the removal of adhesions and fibrotic changes, or targeted administration of medication. According to the literature, in patients with failed back surgery syndrome (FBSS), epidurally applied corticosteroids reach the intended level in only 26% of cases [3]. Anterior epiduroscopy and epiduroscopic laser neural decompression (ELND) have recently been introduced in the treatment of herniated disc decompressions and chronic low back and radicular pain, respectively [2]. Recent publications describe the use of Fogarty catheters and resablation to remove adhesions attached to the dura [3]. There is some controversy regarding the theory behind the role of postoperative fibrosis and epidural adhesions in the etiology of FBSS. This is a syndrome in which patients do not experience an improvement in their clinical status after successful back surgery or, after a minor improvement, their status deteriorates without any correlations with MRI. The formation of scar tissue near the nerve root is a common occurrence after back surgery and is called epidural fibrosis: Scar tissue might be a major cause of postoperative pain, commonly called FBSS. This epidural scarring can cause pain for many reasons; for example, the nerves may be trapped by scars, while veins in the epidural space press down upon the nerves and become enlarged, putting pressure on them [5]. The decreased nourishment of nerve tissue and traction of epidural adhesions on the dural sac can also contribute to the etiopathogenesis of the complex pain syndrome in FBSS. Stenosis of the spinal canal by fibrosis is also a justifiable factor in the worsening of the clinical prognosis in a patient after back surgery. If the aforementioned statements do have a clinical foundation, the removal of fibrosis and adhesions should lead to an improvement in the clinical state of the patient even without the administration of anti-inflammatory medications, depomedrol, and hyaluronidase. On the other hand, it has been scientifically proven that hyaluronidase inhibits cellular recruitment, edema formation, and pro-inflammatory mediator production, resulting in decreased adherence of leukocytes to blood vessels and tissue infiltration [6]. The goals of our study are to evaluate the changes in the clinical state of patients with FBSS after the endoscopic removal of fibrosis and adhesions and to compare between an exclusively mechanical intervention and a mechanical intervention with targeted administration of depot corticosteroids and hyaluronidase. Methods A randomized controlled trial with a parallel group study design was used. The present study was approved by the Ethics Committee of Louis Pasteur University Hospital in Kosice (approval number 75/EK/15) and registered at clinicaltrials.gov with registration ID NCT02459392. Written informed consent was obtained from all participants. All patients with failed back surgery syndrome who were indicated to undergo an epiduroscopy procedure were recruited from one of the three pain clinics in Bratislava, Bardejov, and Kosice in the Slovak republic. Inclusion criteria were age 18years or older, written informed consent, symptoms of FBSS, permanent low back pain with dominant (more than 60%) radiation to lower extremities despite previous epidural corticosteroid injections, current magnetic resonance imaging (no older than three months) without serious spinal stenosis and serious radicular compression. Exclusion criteria were the presence of annihilating phenomena (loss of sensitivity of the skin, loss of coordination of the lower extremities, problems with urination 1437 124 Rapčan et al. Patients approached (N = 89) Excluded (N =44) Randomized (N = 45) Group A mechanical lysis (N = 22) 6-mo follow-up (N: = 22) 12-mo follow-up (N = = 22) Group B mechanical lysis (N = 23) Lost to follow-up (N = l) 6-mo follow-up (N = 22) 12-mo follow-up (N = 22) 5 3 % Figure 1 Flow chart of patient selection, enrollment, and follow-up in the study. or defecation), presence of infection, neoplasms, and lack of patient approval. All eligible patients were approached. After written informed consent was obtained, participants were allocated into study groups according to computer randomization software. Each patient had obtained a unique clinical trial ID number, which was generated by computer software before epiduroscopy. Blinding at the time of randomization was maintained with a sealed envelope given to the anesthesiologist managing the patient. The anesthesiologist was not involved in data collection. Study outcome measurements were obtained by an independent research team from the Medical Faculty, Pavol Jozef Šafárik University in Košice. Study continuance was maintained by an independent clinical study coordinator from the East Slovak Institute of Cardiovascular Disease in Košice, and the report was prepared in accordance with the Consolidated Standards of Reporting Trials (the "CONSORT statement"). All data were assembled in a protected and encrypted database accessible only to the study coordinator including statisticians from an independent statistical institution and local study site coordinators. Patients diagnosed with failed back surgery syndrome (FBSS) were enrolled in the study. Patients underwent at least one previous back surgery with ongoing pain radiating to the lower extremities with or without back pain, which was still present after periradicular therapy or caudal blockade (less than 50% visual analog scale [VAS] relief of pain 30 days after intervention) performed by a pain management specialist. A patient who had met the inclusion criteria was informed by the examining doctor about the study and given the opportunity to participate. They were informed about the intervention procedure, epiduroscopy performed by a flexible fiberoptic endoscope with a video-guided catheter (Myelotec, Inc., Roswell, GA, USA), strictly within the ventral and ventro-lateral epidural space with focus on 1438 125 Drug Administration During Epiduroscopy releasing foraminal adhesions. Consequently, in the case of agreement, the patient signed the informed consent documents about the interventional procedure, epiduroscopy, and their informed consent to participate in the study. The mechanical adhesiolysis itself was performed by three different tools: laser, radiofrequency probe, or a balloon catheter. The choice of the instrument was made by the surgeon according to his clinical preference. The total volume of our standard pharmacological mixture was 30 ml_ per foraminal level (20 mL of the mixture bupivacaine 0.5%, 5ml_ methylprednisolone, 80 mg saline, and 150 I.U. of hyaluronidase, Hylase "Dessau", in 10mL saline). The maximum volume injected was never more than 60 mL. Patients were randomly split into two groups (Figure 1). The first group (Group A) underwent epiduroscopy (5mL of 0.5% bupivacaine was injected; the total volume injected was supplemented up to 20 mL with saline), during which only a mechanical lysis of the epidural fibrotic attachments was performed by either laser (four patients), radiofrequency (15 patients), or the balloon technique (three patients). The second group (Group B) underwent epiduroscopy, during which mechanical lysis of the epidural fibrotic attachments was performed by laser (five patients), radiofrequency (16 patients), or the balloon technique (four patients), as in group A (5mL of 0.5% bupivacaine). At the same time, a solution of hyaluronidase (Hylase "Dessau" 150 I.U. in 10mL of saline) and injectable corticosteroid methylprednisolone acetate (DEPO-Medrol) 80 mg were administered to the patient into the place of conflict (the depression in the spinal root by fibrosis). After completing the first protocol of the preoperative examination, this protocol was sent to the coordinator of the study as well as the researcher in charge of processing study data. The coordinator of the study planned the first postoperative examination six months after the procedure and the second postoperative examination after 12 months following the procedure. The first and second postoperative examinations of the patient were performed by a different physician (not the one performing the actual procedure), or at a different pain management clinic. They performed a pain assessment of the patient while blinded to which procedure they had undergone (endoscopy only including mechanical lysis or with the administration of the drugs) and completed the pain management protocol of the study. Primary outcomes were pain intensity spreading in the back and legs and also evaluation of the Oswestry Disability Index (ODI). Patient Status Score (PSS) is a grading scale from 0-4, where 0 means the patient is without pain, has a normal life, normal job, is able to exercise; and 4 means the patient needs help to take care of themselves and is bed-ridden. The Patient Self-Content Score (PSCS) is evaluated on a scale from 0 to 10, where the patient describes his satisfaction with the procedure at six- and 12-month follow-up (0 being the worst and 10 being the best). There were no important changes to the methods after the study commenced. Table 1 Characteristics of patients in groups divided according to the performed procedure Group A (Min-Max) Med Group B (Min-Max) Med Participants, No. Before procedure 22 6-mo follow-up 22 12-mo follow-up 22 Age, y (35-70) 54 Sex (F/M) 10/12 ASA (1-3) 2 BMI 22 Pain in dermatomes according to examination before procedure L2 0 L3-L4 1 L4-L5 4 L5 2 L5-S1 5 S1 4 Mechanical therapeutic intervention Balloon 3 4 Laser 4 5 Radiofrequency 15 16 23 22 22 (33-69) 46.5 12/11 (1-3) 2 20 Descriptive statistical methods were used to evaluate the results (mean, median, maximum, minimum, and SD). Examination of the distributional form for score and time data was determined by box plots. Each box plot indicated minimal value, lower quartile (lowest 25% of data), median, upper quartile (highest 25% of data), and maximal value. Normality of data distribution was assessed by the Shapiro-Wilk test. Homogeneity of variances was estimated using the Levene test. Differences between continuous variables were analyzed by a non-parametric Kruskal-Wallis one-way test. A paired Student f test was used to assess the statistical significance of changes within each treatment group. P values of less than 0.05 were considered significant. Statistical analysis was performed with the SPSS version 11.0 statistical software i Results Of the 86 admitted patients with FBSS, 45 fulfilled the selection criteria and were randomized into two groups (Group A—mechanical lysis, Group F3—mechanical lysis and drugs) and then underwent epiduroscopy. One patient from group B was lost during follow-up. There were no unexpected side effects. The baseline demographic and characteristic values are summarized in Table 1 and were similar in both groups. A significant improvement was recorded in ODI in both groups after six months (P<0.05), which indicated 1439 126 Rapcan et al. Before procedure Figure 2 Evaluation of patient functional disability by the Oswestry Disability Index indicating significant improvement in comparison with baseline after six months, with return to baseline values after 12 months in both groups. Figure 3 Evaluation of back pain intensity in patients indicating significant reduction in low back pain in both groups after six months, with persisting significant improvement after 12 months in group B. that subjects had greater clinical improvement. After 12 months, the ODI score was the same as before the procedure, which showed a return to the previous state (Figure 2). A reduction in low back pain was recorded after six months in both groups (P>0.05), but this did not persist until 12 months in Group A (P>0.05) (Figure 3). A similar reduction in pain after six months was found on examination of leg pain (P<0.05), but the level of pain in the legs had reverted to baseline after 12 months in both groups (P>0.05) (Figure 4). The changes between both groups are summarized in Table 2. No difference was found between Group A and Group B in ODI scoring values nor in the numerical pain scale for leg pain and back pain before the procedure (TO) or at six months (T1) and 12 months (T2) after procedure (P > 0.05) in all observed parameters. There was no difference in PSS between Group A and Group B. We found significant worsening of PSCS in Group A at P< 0.05, but not in Group B. Discussion Everything we do regarding success or failure in pain medicine is ultimately expressed at the cellular level and represents changes in electrical patterns, neurotransmitters, and metabolism. Epidural fibrosis has been described as a common phenomenon with a place among the major causes of continued pain after surgical intervention [7]. As Baber and Erdek [8] pointed out, scar formation is part of the healing process after spinal surgery, like any other surgical procedure, resulting in fibrosis within the epidural space. Epidural fibrosis can be 1440 127 Drug Administration During Epiduroscopy Before procedure Figure 4 Evaluation of leg pain intensity in patients indicating significant reduction in leg pain in both groups after six months. Table 2 Comparison of the mean values of recorded parameters between groups in the 6 and 12 months following the procedure Parameters Time Interval Group A SD Group B SD Mean 95% CI P Value (Min-Max) (Min-Max) Difference Med Med Oswestry Disability Before procedure (31-80) 65 15.57 (20-76) 58 18.46 -5.192 -15.485 to 5.101 0.313 Index 6-mo follow-up (12-76) 47 18.63 (8-76) 38 21.77 -8.921 -21.090 to 3.248 0.148 12-mo follow-up (18-82) 54 17.78 (12-74) 48 19.14 -8.591 -19.833 to 2.651 0.131 Numerical pain Before procedure (4-9) 7 1.64 (0-10) 8 2.19 1.022 -0.150 to 2.194 0.086 scale- back pain 6-mo follow-up (2-8) 5 1.97 (0-10) 6 2.85 0.561 -0.921 to 2.043 0.449 12-mo follow-up (4-9) 7 1.63 (1-10) 6 2.33 -0.727 -1.951 to 0.496 0.237 Numerical pain Before procedure (3-8) 6 1.72 (4-10) 7 1.54 0.868 -0.071 to 1.806 0.069 scale-leg pain 6-mo follow-up (1-7) 5 1.56 (0-9) 6 2.78 0.887 -0.476 to 2.251 0.193 12-mo follow-up (3-8) 6 1.54 (1-9) 6 2.11 -0.045 -1.254 to 1.163 0.940 Patient status score Before procedure (1-4)3 0.728 (1-4)3 0.700 0.197 -0.239 to 0.632 0.367 6-mo follow-up (0-4) 2 0.976 (0-3) 2 0.839 -0.47 -0.604 to 0.632 0.864 12-mo follow-up (1-4)2 0.728 (1-4)3 1.071 -0.054 -0.620 to 0.512 0.848 Patient self-content 6-mo follow-up (0-10) 7 3.171 (0-10) 7 2.864 0.329 -1.583 to 2.240 0.73 Score 12-mo follow-up (0-10) 5 3.092 (0-10) 6 4.025 1.560 -0.722 to 3.841 0.171 CI = confidence interval. caused by chronic inflammatory changes also from chronic spinal cord injury. The pathophysiological background of epidural fibrosis is the inflammatory reaction of the arachnoid mater [9], The pain is a characteristic manifestation of inflammation there [10]; however, fibrotic adhesions themselves cause back and leg pain by compressing nerve roots, decreasing the range of motion in the back and introducing pain with movement [11]. Despite multiple studies, the relationship between fibrosis and pain is still not entirely resolved [12]. In earlier studies, active signs of root inflammation were seen in only six patients from the 20 studied [13], or none [7]. Therefore, we did not focus on following the signs or markers of inflammation in fibrosis during epiduroscopy. This study concerns an initial evaluation of an initial group of patients over a one-year period after a spinal endoscopic procedure. In our study, an attempt was made to show that the targeted destruction of postoperative fibrosis and adhesions has the potential to improve the patient's clinical condition in cases of 1441 128 O^-^t-COt-CDt-COuD Ot-^t-cO(C^CD^O oooddddoczJd o y id o X m en cd co co r: lo >N cd cd $2 co ^ in o o ^ t- i-CDCNJ^CDLDO-r-T-O cocsjcocncocDcoi-com COr;r;Sr;qrr;nr coroincoscocococ&iri £ * b ^ "5 cm co t- H o o o ffi *" te n w 5S o o P o o o o o o d °* in cm N co co co i— cd CM CD CO N O O CM CO c\i oi I I o o o o o o CO h~ O t— O O N CO CM O CM O CO CO T- i- CO d d d d -r^ ^o>oco^gS OCO^J^J^^OO CO(DO)lOO)COCOC\ISO i-OJOCDUO'tCTiCOinO) cTidiniri'tLri^wcdcD CD N CD CO CM CM Y) the estimated confidence interval covers the expected value 0.5 with stated probability. Otherwise, if the estimated confidence interval does not cover the value 0.5, it was concluded that the alternative is to be preferred. In a nonparametric set-up, the estimator of the reliability flSpri EQ-5D-5L questionnaire as suitable assessment of quality of life after epiduroscopy 134 original article Table 2 Patients' answers distribution in percentage Level/time (months) Group A I II III IV V II III IV V Mobility 0 0.0 47.5 42.5 10.0 0.0 0.0 43.60 51.30 5.10 0.0 6 5.0 52.5 32.5 10.0 0.0 10.50 39.50 26.30 23.70 0.0 12 2.5 42.5 45.0 10.0 0.0 7.80 21.0 41.0 30.20 0.0 Self care 5.0 22.5 62.5 10.0 0.0 8.7 20.5 67.8 3.0 0.0 6 10.0 35.0 47.5 5.0 2.5 10.5 36.8 447 5.4 2.6 12 7.5 22.5 55.0 12.5 2.5 15.8 18.4 55.3 7.9 2.6 Usual activities 0 0.0 17.5 75.0 7.5 0.0 0.0 17.9 71.8 10.3 0.0 6 5.0 35.0 47.5 12.5 0.0 7.9 34.2 50.0 7.9 0.0 12 2.5 5.0 77.5 12.5 2.5 5.0 34.2 52.9 7.9 0.0 Pain/ discomfort 0 0.0 12.5 67.5 17.5 2.5 0.0 11.9 77.9 5.1 5.1 6 5.0 17.5 50.0 20.0 7.5 5.2 23.7 63.2 7.9 0.0 12 2.5 7.5 75.0 10.0 5.0 7.7 23.7 62.2 3.2 3.2 Anxiety/ depression 0 17.5 75.0 5.0 2.5 0.0 7.7 69.0 23.3 0.0 0.0 6 15.0 77.5 7.5 0.0 0.0 8.9 87.5 3.6 0.0 0.0 12 25.0 50.0 20.0 5.0 0.0 29.0 63.5 7.5 0.0 0.0 parameter R is functionally related to the Wilcoxon rank-sum test statistic. For more details see Kotz et al. [91 and Zhou [10]. Analyses using MATLAB (R2018b) with Statistics and Machine Learning Toolbox Version 11.4 (The Math Works, Inc., Natick, MA, USA) were performed. A p-value less than 0.05 was considered statistically significant. For analysis of the Quality of Life (QoL) questionnaire data, Shapiro-Wilk, Levene, and paired t-test, using SPSS Version 11.0 statistic software package, were performed. P values of less than 0.05 were considered significant. Results The following notation was used for the specific groups of patients: AO, A6, A12, B0, B6, B12. Two basic groups of patients (A patients with mechanical lysis only and B patients with mechanical lysis joined with drug administration) were considered. Each group of patients was observed at three different time periods (0 at the beginning of the study, 6 after 6 months from the beginning of the study, and 12 after 12 months from the beginning of the study). The responses of the patients of the two groups to the 5-dimensional EQ-5D-5L questionnaire at all three times are summarized in Table 2. The effectiveness of epiduroscopic treatment in patients with failed back syndrome was assessed by testing statistical hypotheses on equality of probability distributions of the questionnaire responses in the considered (sub) groups of patients against the pre-specified one-sided alternative hypotheses. The hypotheses assume that the distribution of response levels is stochastically smaller, with better health status for one group that the other, in terms of mobility, self-care, usual activities, pain/discomfort and anxiety/depression as defined in 5 dimensional EQ-5D-5L questionnaire. Moreover, as a summary compound measure, we have also considered the comparisons of the combined (weighted) questionnaire values, with carefully prespecified weighing for each questionnaire dimension, which combines the information from all 5 dimensions of the EQ-5D-5L questionnaire. For each subject the combined value Q was calculated by the following formula: Q = 1 - wg - w\ - W2 - 1113 -if4 - W5, with the weights specified in the Table 3. For more details on the different weighing strategies see e.g. Szende et al. [11]. For specific sub-groups of patients, denoted by AO, A6, A12, B0, B6, and B12, we tested the hypotheses on pairwise comparisons. For comparison of any two groups of patients based on the observed questionnaire responses we computed the p-value from the Wilcoxon rank-sum test for testing the null hypothesis of equality of distributions of the questionnaire responses against the (specified) one-sided alternative hypothesis. Moreover, we also estimated the reliability parameter R together with the associated two-sided 95% confidence interval, calculated from the bootstrap distribution of the responses in group 1 and in group 2 (in this study we have used the bootstrap samples of size N = 1000 ). Table 4 presents the observed p-values of the Wilcoxon rank-sum test and the estimated reliability parameter R with the 95% confidence interval for each considered hypothesis on equality of probability distributions of the questionnaire responses in the considered groups of patients. For example, the notation B6< A6 indicates testing the null hypothesis about equality of the distributions for groups B6 and A6, i.e. HO: B6 - A6, against the alternative hypothesis HA: B6< A6 (i.e. better health status in the considered dimension for the patients in group B6 than in the group A6). As is shown in Table 4, there was improvement in mobility after 6 months with statistical significance at p-0.0637 and after 12 months (p<0.05) in EQ-5D-5L questionnaire as suitable assessment of quality of life after epiduroscopy Ö Sprii ger 135 original article ■ Table 3 The weights used for computing the weighted questionnaire value Q, which combines the information from all 5 dimensions of the EQ-5D-5L questionnaire Weight Value EQ-5D-5L dimension Condition WO 0.1279 All Any level in questionnaire is II or III Wo 0.2288 All Any level in questionnaire is IV or V Wo 0.0000 All Otherwise W1 0.0659 Mobility Mobility level is II or III W1 0.1829 Mobility Mobility level is IVorV Wl 0.0000 Mobility Otherwise W2 0.1173 Self-care Self-care level is II or III W2 0.1559 Self-care Self-care level is IV or V W2 0.0000 Self-care Otherwise W3 0.0264 Usual activities Usual activities level is II or III W3 0.0860 Usual activities Usual activities level is IV or V W3 0.0000 Usual activities Otherwise W4 0.0930 Pain/discomfort Pain/discomfort level is II or III W4 0.1639 Pain/discomfort Pain/discomfort level is IV or V W4 0.0000 Pain/discomfort Otherwise W5 0.0891 Anxiety/depression Anxiety/depression level is II or III m 0.1290 Anxiety/depression Anxiety/depression level is IV or V W5 0.0000 Anxiety/depression Otherwise For each subject the value Q was calculated by the formula C weights != 1-W0-W1-W2-W3-W4-W5, with the specified group A. In group B, we recorded a statistically significant improvement in mobility after 6 and 12 months (p<0.05). There was a significant improvement in the performance of normal daily activities in patients, in all groups A and B after 6 and 12 months (p<0.05). Following the pain parameter, group B included patients with significantly smaller pain AO vs. B0 (p<0.05). Subsequently, the reduction in pain was recorded only in A6 and A12 groups (p<0.05) during further measurements. In groups B6 and B12 this trend was not recorded; however, the differences in the pain parameter between A and B after 6 and 12 months were not recorded. Improvement in self-care was observed only in group A after 6 months (p<0.05). After 12 months no improvement in self-care was observed in either group compared to preoperative baseline. An improvement in the sense of anxiety and depression in a given disease varied in both groups. Significant improvements were seen in group A and B after 6 and 12 months (p<0.05). The statistical weighting of values, based on index demographic conversion, showed improvement in group B after 6 and 12 months and group A after 12 months (p<0.05). Normal distribution of EQ-VAS on the numerical scale (0-100) was evaluated with the Shapiro-Wilk test. Homogeneity of variances was estimated using the Levene test. When conditions of homogeneity and normality of variances were met between both groups, for comparing the means we used paired t-test (Table 5, Fig. 2). The comparison shows that significant changes were found in both groups at the 6-month period measurement following the procedure. Interpretation of previous medications in all included patients was extremely difficult and not suitable for statistical evaluation due to the irregular drugs intake, different dosing in all drugs, and high variability of dosing per day even in a single patient. Some of the patients, despite strong pain, did not use any opioids because of opioid intolerance occurrence, which was noticed in around 60% (Fig. 3 and 4). Discussion Chronic pain especially related to FBSS is very difficult to assess properly, as it affects the life of individuals on various levels. Typically used is the measure of pain in the form of visual analogue pain score (VAS), Oswestry disability index and patient's status score as appropriate parameters of dynamic changes in the patient health; however, it could have some limitations. Visual analogue pain score seems to be prone to bias and therefore not very suitable for solitary assessment of these conditions [12]. Neuropathic pain is a complex problem relating to pain centralization in the central nervous system, changes in pain perception, long-term suffering and subsequent behavioral changes [13]. Use of different outcome domains or combination of VAS and another multilevel tool is recommended [14] to assess this condition. Another suitable complementary description method of global patient health evolution after epiduroscopic intervention is EQ-5D questionnaire. It is not specific to one disease, available for self-completion, easy to complete and covers all important aspects of chronic pain states. The longer 5-level version was used, as it pro- flSpri EQ-5D-5L questionnaire as suitable assessment of quality of life after epiduroscopy 136 original article Table 4 Mobility (left) vs. self-care (right) p-values, usual activities (left) vs. pain/discomfort (right) p-values, anxiety/ depression (left) vs. weighted questionnaire (right) p-values of the Wilcoxon ranksum test for testing the null hypothesis of equality of distributions of the questionnaire responses against the specified one-sided alternative, and the estimated reliability Hypothesis p-value R Lower Upper Hypothesis p-value R Lower Upper Mobility Self-care A64 - w5i with the weights specified in Table 3. For more details on the different weighing strategies and for discussion on selecting the specific weights see, for example, Rapcan et al,II2) and Szende.113' Table 4 shows the calculated P values of the Wilcoxon rank-sum test and the estimated values of the reliability parameter R with the associated 2-sided 95% CI. These results are presented for all hypotheses about the equality of the probability distributions of the questionnaire responses in the patient groups considered for each of the 5 dimensions of the EQ-5D-5L questionnaire, that is mobility, self-care, everyday activities, pain/ discomfort, anxiety/depression, and the combined weighted questionnaire score. The Wilcoxon rank-sum test convincingly rejected all considered null hypotheses of equality of the medians of the distributions of the questionnaire responses before and after treatment against the stated 1-sided alternative, which states that the respective dimension of the EQ-5D-5L questionnaire response is better after treatment than before treatment (After < Before). Furthermore, the estimated values of the reliability parameters and the corresponding 95% CIs show that the calculated values for all considered EQ-5D-5L dimensions are higher than 0.5, which clearly confirms the results of the statistical tests. 4. Discussion This study aimed to evaluate clinical parameters, including pain intensity, ODI, and EQ-5D-5L scores, in patients with acute sciatica pain before and after endoscopic discectomy in the Slovak and Czech Republic. The study aimed to test the comprehensibility and face validity of EQ-5D-5L scores compared to other health measurement tools such as the ODI and NRSs for pain. For all patients with LHD in the clinical trial, conservative therapy was initiated after the onset of clinical symptoms for 6 to 8 weeks. This included a combination of steroids, nonsteroidal anti-inflammatory drugs, physical therapy, epidural steroid injections, and rest. If the clinical state did not improve or worsened over time, the treatment strategy was changed to an endoscopic surgical approach. Although there are no proven differences in outcome between conservative and surgical treatment, timely neurosurgical treatment can lead to good long-term results. Nerve decompression performed at the right time is a good prognostic factor for neuronal recovery and can help prevent further irreversible neuronal damage and pain chronification.114-151 Among the monitored patients, the majority had a single-level herniated intervertebral disc in the intervertebral spaces L5/S1 (108 patients) and L4/L5 (169 patients). We observed a persistent improvement in the perception of back pain and pain radiating to the lower limbs, with a significant improvement in quality of life 1 year after the follow-up as measured by the EQ-VAS visual scale and the Oswestry disability questionnaire. These findings are consistent with (he results <>t other clinical trials using various surgical approaches for lumbar herniated ilises. A systematic review and meta-analysis of 99 articles focused on various spinal conditions found that spine surgery was associated with improved HRQoE for all groups in which postoperative scores were measured. The analysis included 22,312 cases for EQ-5D utilities, 2312 cases for SF-6D utilities, and 11,927 cases for SF-36 PCS scores with a median follow-up time of 12 months. Clinical trials monitoring the treatment of lumbar herniated discs with open surgical approaches, such as hcmilaminectomy and microdiscectomy, have also shown the benefit of the procedures and improved patient quality of life after surgery. However, open surgery is associated with disadvantages such as extensive retraction and dissection of paraspi-nal muscles, longer operating time, larger wounds, and bone resection.1"11"1 Minimally invasive surgical approaches offer several advantages over open surgery, including the preservation of paraspinal structures and a reduction in postoperative pain, which often leads to earlier discharge. Additionally, these procedures can be performed under local anesthesia. A meta-analysis was conducted to compare the effectiveness of open lumbar surgery with lumbar endoscopic discectomy. The analysis included 9 randomized controlled trials with 1092 patients. The results showed that endoscopic surgery had slightly better clinical outcomes than open surgery based on 152 Rapcanetal. • Medicine (2023) 102:26 www.md-journal.com 5 i low back pain leg pain low back pain leg pain before procedure 1 year follow-up Figure 2. Evaluation of patients' scores on the Numerical Pain Scale (NRS) for lower limb and back pain, before treatment, and 12 months after treatment. T ODI EQ-VAS before procedure ODI EQ-VAS 1 year follow-up Figure 3. Evaluation of patients' scores of Oswestry Disability Index (ODI) and overall health score (EQ-VAS) before treatment, and 12 months after treatment. Numerical scafe (0-100) of ODI and EQ-VAS before the procedure and at the 1 wear follow-up. the Macnab criteria, but this difference was not clinically significant. However, patients who underwent endoscopic surgery reported significantly higher satisfaction rates, lower intraoperative blood loss volume, and shorter hospital stays.161 Similarly, a meta-analysis compared the efficacy of PELD with other surgeries for LDH and demonstrated better outcomes of PELD in 14 clinical trials involving a total of 2528 patients. PELD had a shorter duration of surgery, lower blood loss, and similar complications compared to orher surgeries for LDH. However, PELD operations resulted in a higher recurrence rate (relative risk = 1.65, 95% CI: 1.08-2.52; P = .021). There were no significant differences in quality of life and ODI before and after the procedure between the interventions. The study's limitation was the relatively low number of clinical trials included.1,1 Nevertheless, published results concerning open-operative techniques are also associated with a good prognosis. To evaluate the quality of life, we chose the EQ-5D-5L questionnaire as it offers a comprehensive assessment of a patient's health status. This questionnaire measures 5 dimensions of perceived problems, each with 5 levels of severity, which improves sensitivity and reduces the ceiling effect. The EQ-VAS scale, which is easier to complete and score, was also used. The "Usual activities" dimension evaluates a patient's performance in work, studying, housework, family, or leisure activities, as well as other dimensions like mobility, self-care, everyday activities, and anxiety/depression. Our analysis shows 153 Rapčanetal. • Medicine (2023) 102:26 Medicine Table 3 The weights used for computing the weighted questionnaire value Q, which combines the information from all 5 dimensions of the EQ-5D-5L questionnaire. Weight Value EQ-5D-5L dimension Condition wO 0.1279 All Any level in questionnaire is 2 or 3 wO 0.2288 All Any level in questionnaire is 4 or 5 wO 0.0000 All Otherwise w1 0.0659 Mobility Mobility level is 2 or 3 w1 0.1829 Mobility Mobility level is 4 or 5 w1 0.0000 Mobility Otherwise w2 0.1173 Self-care Self-care level is 2 or 3 w2 0.1559 Self-care Self-care level is 4 or 5 tv? 0.0000 Self-care Otherwise w3 0.0264 Usual activities Usual activities level is 2 or 3 w3 0.0860 Usual activities Usual activities is 4 or 5 w3 0.0000 Usual activities Otherwise w4 0.0930 Pain/discomfort Pain/discomfort level is 2 or 3 w4 0.1639 Pain/discomfort Pain/discomfort level is 4 or 5 w4 0.0000 Pain/discomfort Otherwise w5 0.0891 Anxiety/depression Anxiety/depression level is 2 or 3 w5 0.1290 Anxiety/depression Anxiety/depression level is 4 or 5 w5 0.0000 Anxiety/depression Otherwise For each subject, the value 0 was calculated by the formula Q = -\-w0-Wi-W2-w3-Wt-w>, with the specified weights. Calculated P values of the Wilcoxon rank-sum test and the estimated values of the reliability parameter R. Wilcoxon EQ-5D-5L Alternative test P Reliability parameter R dimension hypothesis value Prob (After < Before) Lower Upper Mobility After1 year after endoscopic surgery (P < .001). We also calculated the statistically weighted questionnaire values for each dimension of the EQ-5D-5L questionnaire in the Slovak and Czech region, which also indicate a significant improvement in social quality of life (P< .001). Despite our study involving patients with a mean age of 46.3 years, the clinical improvement, reduction in lower limb pain, and improved quality of life after surgery are consistent with a 2-year follow-up study by Peng et al[(S| in younger patients with a mean age of 35.6 years. An interesting finding from a prospective cohort study by Kapetanakis et alH1 is that neither gender, muscle mass, nor body mass index has a significant effect on the final outcome of endoscopic discectomy. A systematic analysis of clinical trials comparing the effectiveness of transforaminal and interlaminar endoscopic approaches in treating LHD, which reviewed 26 clinical trials involving 3294 patients, demonstrated a significant therapeutic benefit with both interventional approaches. However, the transforaminal approach was associated with higher efficacy, shorter operation time, and lower blood loss.17' In our clinical study, we selected the safest and most effective possible approach for each patient. For patients with higher levels of L3/4 and L4/5 herniations, we preferred the transforaminal approach. For herniations in the lower spinal segments L5/S1 due to anatomical conditions of the pelvis, we chose the interlaminar approach more often than the transforaminal approach. Comparing both approaches, we did not observe a higher percentage of complications or re-herniation. We consider analgosedation with monitoring as a safe and beneficial method for endoscopic discectomy. It allows for effective communication with the patient, ensuring safety and minimizing the risk of nerve damage. Additionally, it promotes early recovery and a faster return to consciousness, while reducing the occurrence of postoperative nausea and vomiting. Despite potential challenges in airway access, we did not encounter any cases of acute respiratory failure requiring intubation. 4.1. Limitations Our clinical trial on acute sciatica treatment had limitations that should be addressed. Firstly, using a prospective observational design instead of a randomized controlled trial introduced the potential for selection bias. The absence of a control group made it challenging to compare treatment effectiveness accurately. Including a control group would have enabled a more robust evaluation of endoscopic discectomy, microdiscectomy, and conservative treatments. The study involved 470 patients, but a broader age range (18-79 years) may have influenced the results. Additionally, a loss to follow-up of 39 patients resulted in missing data for statistical analysis, potentially impacting the robustness and gener-alizability of our findings. Future studies should minimize loss to follow-up and ensure comprehensive data collection. Recognizing these limitations is crucial for interpreting our findings accurately and planning future research in acute sciatica treatment. 5. Conclusions Endoscopic discectomy is a highly effective minimally invasive surgical method for treating LHD, with a significant impact on the patient's clinical state. Clinical assessments of the quality of life using different types of measurement instruments are useful in demonstrating the efficacy of pain-treating interventions. Author contributions Conceptualization: Ladislav Kočan,Juraj Mláka, RóbertTirpák. Data curation: Róbert Rapčan, Ladislav Kočan, Viktor Witkovsky. Formal analysis: Róbert Rapčan, Ladislav Kočan, Viktor Witkovsky, Miroslav Gajdoš. Funding acquisition: Ladislav Kočan, Simona Rapčanová, Juraj Mláka. Investigation: Ladislav KoČan, Viktor Witkovsky, Simona Rapčanová, Juraj Mláka, RóbertTirpák, Hana Kočanová. Methodology: Ladislav Kočan, Hana Kočanová. Project administration: Ladislav Kočan, Janka Vašková. Resources: Ladislav Kočan, Miroslav Burianek, Janka Vašková. Software: Róbert Rapčan. Supervision: Róbert Rapčan, Ladislav Kočan, Juraj Mláka. Validation: Róbert Rapčan, Ladislav Kočan, Miroslav Burianek. Visualization: Miroslav Burianek. Writing - original draft: Ladislav Kočan, Simona Rapčanová, Róbert Tirpák, Hana Kočanová. 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Statistical inference for P(X