Sažetak | Cilj istraživanja: Ispitati stvaranje reaktivnih kisikovih spojeva u ljudskom miokardu nakon
terapijskog postavljanja aorto-koronarne premosnice i mogućnosti njihovog smanjenja.
Materijali i metode: U svrhu usporedbe razine proizvedenih reaktivnih kisikovih vrsta kod
skupine pacijenata koji imaju razvijene koronarne kolateralne krvne žile i skupine koji ih
nemaju, uzeti su uzorci od ukupno 11 pacijenata. Tijekom izvođenja operativnog zahvata
postavljanja srčane premosnice bez upotrebe stroja za izvantjelesnu oksigenaciju krvi (tzv. offpump CABG metoda) sakupljeni su uzorci tkiva subepikardijalnog miokarda lijevog ventrikula
metodom iglene biopsije u dva navrata - prije postavljanja premosnice (dakle u fazi ishemije) i
5 minuta nakon ugradnje premosnice (u fazi reperfuzije). Razvoj koronarnih kolateralnih krvnih
žila se procijenio korištenjem koronarne angiografije, a ocijenio Rentropovim sustavom.
Neposredno nakon uzimanja, uzorci su uronjeni u tekući dušik, što je omogućilo kvantifikaciju
ROS-a koja je bila prisutna in situ u tom trenutku. Potom su napravljena mjerenja metodom
elektronske paramagnetne rezonance (EPR). Da bi se putem EPR-a izmjerila razina ROS-a u
uzorak se dodaje CMH indikator (1-hidroksi-3-metoksikarbonil-2,2,5,5-tetrametilpirolidin)
koji se mijenja u reakciji s ROS-om, pri čemu nastaje stabilni paramagnetski CM• (3-
metoksikarbonil-proksil nitroksid) kojeg detektira EPR uređaj. U podgrupi od 5 pacijenata u
fazi ishemije (prije postavljanja premosnice) je uzet dodatni uzorak tkiva za potrebe analize
proizvodnje ROS-a in vitro. Ti su uzorci odmah uronjeni u kardioplegičnu otopinu te
dopremljeni u laboratorij gdje su se pripremili za eksperiment u uređaju za homogenizaciju
tkiva (u koji je dodana i respiracijska otopina). Tako pripremljeni homogenat je prebačen u
eksperimentalnu komoricu (uređaja za mjerenje mitohondrijskih funkcija) uz dodatak
metaboličkih supstrata i Amplex Red-a (detekcijskog sustava koji omogućuje procjenu
stvaranja ROS-a). Amplex Red veže vodikov peroksid pri čemu se formira produkt rezorufin
koji ima fluorescencijsku sposobnost koju detektiramo u sustavu i iz koje računamo razinu
proizvedenog peroksida.
Rezultati: Metodom EPR-a izmjerena je razina ROS-a na temelju proporcionalnosti
paramagnetnom CM•. Kada su svi pacijenti uključeni u statističku analizu nije pronađena
statistički značajna razlika u porastu stvaranja ROS-a. No, pri analizi skupine pacijenata koji
nemaju razvijene kolateralne krvne žile (Rentrop ocjena = 0) ustanovljeno je da je reperfuzija
dovela do 56% porasta razine ROS-a, P < 0.05. S druge strane, kod pacijenata koji imaju
razvijene kolateralne krvne žile (Rentrop ocjena ≥ 2) nisu utvrđene promjene razine ROS-a. U
metaboličkoj komori za mjerenje mitohondrijske funkcije prvo je izmjerena dinamika stvaranja
ROS-a za vrijeme oksidativne fosforilacije, koja se pokazala relativno niskom.
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Potom je tkivo miokarda izloženo metaboličkim uvjetima simulirane reperfuzije (od kojih su
glavni visokoreducirani ubikvinol i visok protonski gradijent preko unutarnje mitohondrijske
membrane) tijekom koje je proizvodnja ROS-a bila značajno povećana. Kada se uzorak izložio
istim uvjetima simulirane reperfuzije, ali u prisutnosti metformina, proizvodnja ROS-a je bila
značajno niža.
Zaključci: Prilikom reperfuzije ishemičnog tkiva dolazi do porasta proizvodnje ROS-a
mehanizmom reverznog toka elektrona. Porast proizvodnje ROS-a u uvjetima simulirane
reperfuzije se smanjuje primjenom metformina. Kod pacijenata podvrgnutih aortokoronarnom
premoštenju dolazi do značajnog porasta ROS-a u fazi reperfuzije kod one skupine koja nema
razvijene kolateralne krvne žile. Prema tome, otvara se mogućnost korištenja metformina u
svrhu profilakse razvoja reperfuzijske ozljede, idealno prije podvrgavanja pacijenata nekoj od
revaskularizacijskih metoda, od čega će najveću korist imati oni pacijenti koji nemaju razvijenu
koronarnu kolateralnu cirkulaciju. |
Sažetak (engleski) | Research aim: Investigate the generation of reactive oxygen species in human myocardium
after its revascularization by coronary artery bypass graft surgery and probe the possibilites of
their reduction.
Materials and methods: In order to compair the level of reactive oxygen species produced in
the group of patients with developed coronary artery collaterals and the group without them,
samples were taken from a total of 11 patients. During the performance of the off-pump CABG,
tissue samples of the subepicardial myocardium of the left ventricle were collected by the
needle biopsy method on two occasions - before the installation of the bypass (thus in the phase
of ischemia) and 5 minutes later after installation of the bypass (in the reperfusion phase). The
development of coronary collateralls was assessed using coronary angiography, and evaluated
by the Rentrop system. Immediately after collection, the samples were immersed in liquid
nitrogen, which allowed the quantification of ROS present in situ at that time. Measurements
were then made using the electron paramagnetic resonance (EPR) method. In order to measure
the level of ROS via EPR, a CMH indicator (1-hydroxy-3-methoxycarbonyl-2,2,5,5-
tetramethylpyrrolidine) was added to the sample, which changes in the reaction with ROS,
resulting in stable paramagnetic CM• (3-methoxycarbonyl-proxyl nitroxide) detected by EPR
device. In a subgroup of 5 patients in the phase of ischemia (before bypass placement), an
additional tissue sample was taken for the analysis of ROS production in vitro. These samples
were immediately immersed in the cardioplegic solution and delivered to the laboratory where
they were prepared for the experiment in the tissue homogenization device (to which the
respiratory solution was added). The homogenate thus prepared was transferred to an
experimental chamber (in a device for measuring mitochondrial functions) with the addition of
metabolic substrates and the Amplex Red (a detection system that enables the assessment of
ROS formation). Amplex Red binds hydrogen peroxide, forming the product resorufin, which
has a fluorescence ability that we detect in the system and from which we calculate the level of
produced peroxide.
Results: Using the EPR method, the level of ROS was measured based on proportionality with
paramagnetic CM•. There was no difference in ROS between ischemia and reperfusion when
all patients were pooled together. However, in group of patients with no collaterals (Rentrop =
0), reperfusion induced 56% increase in ROS (P < 0.05). In patients with developed collaterals
(Rentrop ≥ 2), no change in ROS levels was found. In the metabolic chamber for measuring
mitochondrial function, the dynamics of ROS generation during oxidative phosphorylation was
first measured, which turned out to be relatively low.
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The myocardial tissue was then exposed to the metabolic conditions of simulated reperfusion
(the main ones being highly reduced ubiquinol and a high proton gradient across the inner
mitochondrial membrane) during which ROS production was significantly increased. When the
sample was exposed to the same conditions of simulated reperfusion, but in the presence of
metformin, ROS production was significantly lower.
Conclusions: During reperfusion of ischemic tissue, there is an increase in the production of
ROS by the mechanism of reverse electron transfer. The increase in ROS production under
conditions of simulated reperfusion is reduced by the administration of metformin. In patients
undergoing CABG, there is a significant increase in ROS in the reperfusion phase in the group
that does not have developed collateral blood vessels. Therefore, the possibility of using
metformin for the purpose of prophylaxis of the development of reperfusion injury opens up,
ideally before subjecting patients to revascularization methods, from which those patients who
do not have developed coronary collateral circulation will benefit the most. |