Absorbance at 593 nm was recorded for 4 min in a microplate reader TECAN (Salzburg, Austria) to determine the rate of Fe(II)-DPP complex formation as compared to a Fe(II) standard curve. Total FRAP was calculated by determining the area under curves within the
time-span of t0 and t60 (AUCt0-t60). Total heme-iron content in plasma Heme-iron content in plasma was assayed with a specific biochemical kit from Doles-Bioquímica Clínica (Brazil). The method is based on the heme-iron oxidation by the ferricyanide anion contained in a solution with 0.10 M potassium dihydrogenophosphate, 60 mM K3[Fe(CN)6, 77 mM Protein Tyrosine Kinase inhibitor KCN and 82 mM Triton X-100). Total heme-iron cyanide – which includes heme groups from hemoglobin, myoglobin, and other hemeproteins – is stoichiometrically detected at 540 nm [28, 29]. Total heme-iron released in plasma was calculated by determining the area under curves within the time-span of t0 and t60 (AUCt0-t60). Uric acid determination Plasma uric acid content was assayed with a biochemical kit from BioClin-Quibasa (Belo Horizonte, Brazil). In the assay mixture, H2O2 produced from uric acid in the presence of uricase (to form allantoin) is coupled with p-hydroxybenzoate and 4-aminoantipyrine oxidation catalyzed by peroxidase to form a pinkish chromophore detected
at λ = 505 nm . Total uric acid released in plasma was calculated by determining the area under curves within the time-span of t0 and t60 (AUCt0-t60). Furthermore, total uric acid https://www.selleckchem.com/products/BKM-120.html released in plasma within the t0 – t60 interval was correlated with total FRAP, but comparison was purposely made with following groups: (i) subject samples not affected by creatine: pre-placebo, post-placebo, and pre-creatine together; and (ii) post-creatine samples. Lipid peroxidation measurements One of the most frequently evaluated byproducts from lipid peroxidation is malondialdehyde Branched chain aminotransferase (MDA), which was accurately analyzed here by chromatographic HPLC technique . The biomarker MDA was first dispersed from
cellular compartments in a mixture of 50 μL of plasma with 250 μL methanol 30 % for 15 min (4o C) in an ultrasound bath. Afterwards, 100 μL of 0.50 % butylated hydroxytoluene (BHT) were added to the samples to avoid oxidation reactions in the following steps. Molecules of MDA were then converted into a pinkish chromophore by derivatization with 600 μL of a 0.4 % thiobarbituric acid solution (TBA, in 0.20 M HCl) for 30 min at 95o C, under constant mixing. The sample was then filtered (MilliPore nylon membranes, 0.45 μm pore size, 13 mm diameter) and injected (20 μL) in a Shimadzu SCL10A HPLC system provided with LC10AD pumps and fluorescence (RF-10AXL) detector. The MDA-TBA adduct was isocratically eluted by the 65:35 mixture of 25 mM phosphate buffer (pH 6.5) and methanol 30 % through a 0.