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Raman spectroscopy is a fast, non-destructive and reliable analytical technique for H2O2 quantification, which avoids the drawbacks of traditional iodometric determinations (sample extraction, preparation of the reagents and a long time of analysis).
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For enzymatic activity determinations, samples were stored into K-3-EDTA tubes at 4 °C for no more than 2 days.
For total eNOS determinations, samples were mixed with Lamelli buffer, heated to 95°C for 5 min, and loaded onto a 7.5% SDS-PAGE and electrophoresis was perform at room temperature.
Animal study sample size determination: sample sizes were determined using the samples size estimation formula for differences in means with power set 80%, two-sided α = 0.05, and a pre-specified effect size of 30%.
Table 1 Results of S BET determination Sample S BET (m2/g) TiNT 540.2 PDA-TiNT 38.4.
For protein determination, samples (4 × of 0.20 g) were digested with 5 ml of mixture H2SO4 + H3PO4 (50:1) with addition of 2.5 ml H2O2 on 420°C.
For mercury determination, samples were diluted 20-fold in a solution containing ammonium hydroxide before analysis.
For each determination, samples containing 5 μg liver protein were used.
For lipid determination, samples were evaporated to dryness after the extraction step, and the amount of lipids was determined gravimetrically.
For CFU determination, samples were diluted and plated (two plates/dilution) on solid Middlebrook 7H10 medium plates, prepared as described above.
After H2O2 determination, samples were washed thoroughly and corrected for cell number using a CytoSelect colormetric assay kit (Cell Biolabs, Inc., San Diego, CA).
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