All blood analyses were performed using a free radical analyzer system (FREE Carpe Diem, Wimerll Company Ltd., Tokyo, Japan) that included a spectrophotometric device reader and a thermostatically regulated mini-centrifuge, and the measurement kits were optimized to the FREE Carpe Diem System, according to the manufacturer's instructions. Based on the recommendation from the manufacturer, all analyses were performed within 48 hours of venous blood collection to avoid falsely high or low results. To analyze the plasma levels of reactive oxygen metabolites, antioxidant capacity, and thiol-antioxidant capacity, diacron reactive oxygen metabolite (dROM), biological antioxidant potential (BAP), and sulfhydryl (SH) tests were performed, respectively.
The dROM test reflects the amount of organic hydroperoxides that is related to the free radicals from which they are formed. When the samples are dissolved in an acidic buffer, the hydroperoxides react with the transition metal (mainly iron) ions liberated from the proteins in the acidic medium and are converted to alkoxy and peroxy radicals. These newly formed radicals oxidize an additive aromatic amine (N,N-diethyl-para-phenylen-diamine) and cause formation of a relatively stable colored cation radical that is spectrophotometrically detectable at 505 nm [33] (link), [36] (link). The results are expressed in arbitrary units (U. Carr), one unit of which corresponds to 0.8 mg/L of hydrogen peroxide [33] (link), [36] (link).
The BAP test provides an estimate of the global antioxidant capacity of blood plasma, measured as its reducing potential against ferric ions. When the sample is added to the colored solution obtained by mixing a ferric chloride solution with a thiocyanate derivative solution, decoloration results. The intensity of the decoloration is spectrophotometrically detectable at 505 nm and is proportional to the ability of plasma to reduce ferric ions [34] (link), [37] (link). The results are expressed in µmol/L of the reduced ferric ions.
The SH test provides an estimate of the total thiol groups in the biologic samples, using a modified Ellman method [38] (link), [39] (link). When the sample is added to the solution, sulfhydryl groups in the sample react with 5,5-dithiobus-2-nitrobenzoic acid, which is followed by development of a stained complex that is spectrophotometrically detectable at 405 nm and is proportional to their concentration according to the Beer-Lambert law [34] (link), [36] (link). The results are expressed as µmol/L of the sulfhydryl groups.
The dROM test reflects the amount of organic hydroperoxides that is related to the free radicals from which they are formed. When the samples are dissolved in an acidic buffer, the hydroperoxides react with the transition metal (mainly iron) ions liberated from the proteins in the acidic medium and are converted to alkoxy and peroxy radicals. These newly formed radicals oxidize an additive aromatic amine (N,N-diethyl-para-phenylen-diamine) and cause formation of a relatively stable colored cation radical that is spectrophotometrically detectable at 505 nm [33] (link), [36] (link). The results are expressed in arbitrary units (U. Carr), one unit of which corresponds to 0.8 mg/L of hydrogen peroxide [33] (link), [36] (link).
The BAP test provides an estimate of the global antioxidant capacity of blood plasma, measured as its reducing potential against ferric ions. When the sample is added to the colored solution obtained by mixing a ferric chloride solution with a thiocyanate derivative solution, decoloration results. The intensity of the decoloration is spectrophotometrically detectable at 505 nm and is proportional to the ability of plasma to reduce ferric ions [34] (link), [37] (link). The results are expressed in µmol/L of the reduced ferric ions.
The SH test provides an estimate of the total thiol groups in the biologic samples, using a modified Ellman method [38] (link), [39] (link). When the sample is added to the solution, sulfhydryl groups in the sample react with 5,5-dithiobus-2-nitrobenzoic acid, which is followed by development of a stained complex that is spectrophotometrically detectable at 405 nm and is proportional to their concentration according to the Beer-Lambert law [34] (link), [36] (link). The results are expressed as µmol/L of the sulfhydryl groups.
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