The determination of the cerium content in the composites was carried out using the UV spectroscopy method. The measurements were carried out on a Specord M40 device from Carl Zeiss (Jena, Germany) in the spectral range from 280 to 500 nm [34 (link),35 (link)]. Sample solutions were prepared to record the UV spectra. A calibration graph was built according to the method given in [15 (link)]. To construct this calibration graph, weighed amounts of cerium ammonium nitrate (0.5 mg, 1 mg, 1.5 mg, and 2 mg) were dissolved in 100 µL of concentrated H2SO4. Then, 10 mL of an aqueous solution containing 0.1% wt. of silver nitrate and 0.2 g of ammonium persulfate was added to the solutions. After that, the UV spectra of the obtained solutions were recorded in the wavelength range from 200 to 500 nm and the absorption intensity was measured at a wavelength of 310 nm (D). Absorption spectra of the solutions containing cerium ions of various concentrations and a calibration curve are presented in Supplementary Materials Figure S1 .
Specord m40
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18 protocols using specord m40
1
Quantitative Analysis of Cerium in Composites
2
Antioxidant Enzyme Activity Assay
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SOD activity was determined using diagnostic kit RANSOD produced by RANDOX (Randox Laboratories Ltd., Crumlin, Country Antrim, UK) according to Arthur and Boyne [19 (link)] and expressed in U of SOD/10mgof protein.
GPx activity was determined using diagnostic kit RANSEL produced by RANDOX (Randox Laboratories Ltd., Crumlin, Country Antrim, UK) according to Paglia and Valentine [20 (link)] and expressed in U of GPx/mg of protein. Protein was measured using method of Bradford [21 (link)]. The assays were performed with the use of spectrophotometer SPECORD M40 (Carl Zeiss, Jena, Germany).
GPx activity was determined using diagnostic kit RANSEL produced by RANDOX (Randox Laboratories Ltd., Crumlin, Country Antrim, UK) according to Paglia and Valentine [20 (link)] and expressed in U of GPx/mg of protein. Protein was measured using method of Bradford [21 (link)]. The assays were performed with the use of spectrophotometer SPECORD M40 (Carl Zeiss, Jena, Germany).
3
Synthesis and Purification of Acrylamide Hydrogels
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The method of homophase radical polymerization in aqueous medium was used for synthesis of hydrogels based on acrylamide [32 ]. Polymerization was carried out at room temperature. The monomer solution was stirred on a laboratory magnetic stirrer (MM-5, 1200 rpm), then the redox initiation system (potassium persulfate–sodium metabisulfite) was added. The components’ concentrations in the initiating mixture were chosen to achieve complete polymerization within 1 h and to prevent excessive composition heating, which could adversely affect the hydrogels’ properties. Cross-linking with the spatial network formation occurred due to copolymerization with a bifunctional monomer, N,N′-methylene-bis-acrylamide (MBA). The gel was dispersed in a mortar to a granule size of Ø1–2 mm. The components used for PAAG synthesis are summarized in Table 1 .
The synthesized PAAGs were repeatedly washed to remove unreacted components of the reaction mixture in distilled water in a ratio of 1 to 50 at a temperature of 45 °C for 7 days. The washing process was monitored spectrophotometrically using a UV spectrophotometer SPECORD M40 (Carl Zeiss) [32 ]. The washing water was further analyzed for the presence of toxic impurities using test objects of plant and animal origin.
The synthesized PAAGs were repeatedly washed to remove unreacted components of the reaction mixture in distilled water in a ratio of 1 to 50 at a temperature of 45 °C for 7 days. The washing process was monitored spectrophotometrically using a UV spectrophotometer SPECORD M40 (Carl Zeiss) [32 ]. The washing water was further analyzed for the presence of toxic impurities using test objects of plant and animal origin.
4
Quantifying Flavonoids in Honey
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Each honey sample (0.5 g) was dissolved in 1 mL of distilled H2O. The honey solution was mixed with 300 μL of 5% NaNO2. After 5 minutes, 300 μL of 10% AlCl3 was added, and the mixture was vortexed. After 6 minutes, the solution was neutralised with the addition of 2 mL of NaOH (1 M). The absorbance was measured at 510 nm against the blank using a spectrophotometer (Specord M40, Carl Zeiss, Jena, Germany). Catechin (1.25–50.0 μg mL−1) was used as the standard, and the total flavonoid content was expressed as catechin equivalents (CAE) (mg CAE/100 g of honey).15 (link)
5
Determination of Total Phenolic Content in Honey
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The Folin–Ciocalteu method described by Ferreira et al. [25 (link)] was used to determine total phenolic content (TPC) with some modifications. Honey sample (1 g) was dissolved in 20 mL of distilled H2O. The honey solution was used (8 mL) and mixed with 500 μL of diluted Folin–Ciocalteu reagents (1:2) for 3 min. Thereafter, 1.5 mL of sodium carbonate (25%) was added. The mixture was shaken and left to stand in the dark at 22 ± 1 °C for 2 h. The absorbance was measured at 750 nm using a spectrophotometer (Specord M40, Carl Zeiss, Jena, Germany). Gallic acid (1.25–31.25 μg/mL) was used as the standard for the construction of the calibration curve, and the TPC was expressed as gallic acid equivalents (GAE) (mg GAE/100 g of honey).
6
Blood Biomarker Evaluation Protocol
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The concentration of TNF-alpha in the blood serum was measured by enzyme immunoassay (enzyme-linked immunosorbent assay) (R&D Systems, Inc., Minneapolis, MN, USA). Total antioxidant status (TAS) was measured using a TAS Randox kit (Randox Laboratories, Ltd, Crumlin, UK) and spectrophotometry (SPECORD M40; Carl Zeiss Jena, Germany). Serum CRP level was determined by ELISA (R&D system, Minneapolis, Minnesota, USA).
7
Oxidant Parameters in Cancer Tissue
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The tissue samples were rinsed with 0.9% NaCl and stored at –80°C until the analysis. Tissue homogenates (10% w/v) were prepared in 0.1 mol l–1Tris-HCl buffer, pH = 7.4 using a laboratory MPW-120 homogenizer, and supernatants were obtained by centrifugation at 5000 × g for 30 min.
The following oxidant parameters were determined in homogenates of cancer tissue: total antioxidant status (TAS) values as well as activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx).
TAS values were assayed using diagnostic kit produced by RANDOX (Randox Laboratories Ltd., Crumlin, County Antrim, UK) according to Miller et al. [23 (link)] and expressed in mmol of TAS/10 mg of protein.
SOD activity was determined using diagnostic kit RANSOD produced by RANDOX (Randox Laboratories Ltd., Crumlin, County Antrim, UK) according to Arthur and Boyne [24 (link)] and expressed in U of SOD/10 mg of protein.
GPx activity was determined using diagnostic kit RANSEL produced by RANDOX (Randox Laboratories Ltd., Crumlin, County Antrim, UK) according to Paglia and Valentine [25 (link)] and expressed in U of GPx/mg of protein. Protein was measured using the method of Bradford [26 (link)]. The assays were performed with the use of spectrophotometer SPECORD M40 (Carl Zeiss, Jena, Germany).
The following oxidant parameters were determined in homogenates of cancer tissue: total antioxidant status (TAS) values as well as activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx).
TAS values were assayed using diagnostic kit produced by RANDOX (Randox Laboratories Ltd., Crumlin, County Antrim, UK) according to Miller et al. [23 (link)] and expressed in mmol of TAS/10 mg of protein.
SOD activity was determined using diagnostic kit RANSOD produced by RANDOX (Randox Laboratories Ltd., Crumlin, County Antrim, UK) according to Arthur and Boyne [24 (link)] and expressed in U of SOD/10 mg of protein.
GPx activity was determined using diagnostic kit RANSEL produced by RANDOX (Randox Laboratories Ltd., Crumlin, County Antrim, UK) according to Paglia and Valentine [25 (link)] and expressed in U of GPx/mg of protein. Protein was measured using the method of Bradford [26 (link)]. The assays were performed with the use of spectrophotometer SPECORD M40 (Carl Zeiss, Jena, Germany).
8
Carotenoid Content Extraction in Honey
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Total carotenoid content (TCC) extraction was carried out using the method previously described by Ferreira et al.14 (link) with some modifications. Two grams of each sample was shaken with 10 mL of n-butanol saturated with H2O. The sample was then left to stand in the dark at room temperature for 18 h. Consequently, the sample was mixed and filtered through a filter paper (Whatman, Grade 4 Chr, UK). The absorbance was measured at 436 nm in comparison to the blank using a spectrophotometer (Specord M40, Carl Zeiss, Jena, Germany). Beta-carotene was used for the construction of the calibration curve (0.24–3.84 μg mL−1). The total carotenoid content was expressed as mg of β-carotene equivalents (BCE) (mg BCE/kg of honey).
9
Oxidant Parameters in Cancer Tissue
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The tissue samples were rinsed with 0.9 % NaCl and stored at −80 °C until the analysis. Tissue homogenates (10 % w/v) were prepared in 0.1 mol. l–1Tris-HCl buffer, pH = 7.4 using a laboratory MPW-120 homogenizer, and supernatants were obtained by centrifugation at 5000× g for 30 min.
The following oxidant parameters were determined in homogenates of cancer tissue: activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx).
SOD activity was determined using diagnostic kit RANSOD produced by RANDOX (Randox Laboratories Ltd., Crumlin, County Antrim, UK) according to Arthur and Boyne [23 (link)] and expressed in U of SOD/10 mg of protein.
GPx activity was determined using diagnostic kit RANSEL produced by RANDOX (Randox Laboratories Ltd., Crumlin, County Antrim, UK) according to Paglia and Valentine [24 (link)] and expressed in U of GPx/mg of protein. Protein was measured using the Bradford method [25 (link)]. The assays were performed with the use of spectrophotometer SPECORD M40 (Carl Zeiss, Jena, Germany).
The following oxidant parameters were determined in homogenates of cancer tissue: activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx).
SOD activity was determined using diagnostic kit RANSOD produced by RANDOX (Randox Laboratories Ltd., Crumlin, County Antrim, UK) according to Arthur and Boyne [23 (link)] and expressed in U of SOD/10 mg of protein.
GPx activity was determined using diagnostic kit RANSEL produced by RANDOX (Randox Laboratories Ltd., Crumlin, County Antrim, UK) according to Paglia and Valentine [24 (link)] and expressed in U of GPx/mg of protein. Protein was measured using the Bradford method [25 (link)]. The assays were performed with the use of spectrophotometer SPECORD M40 (Carl Zeiss, Jena, Germany).
10
Oxidative Stress Markers in Surgical Patients
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Ulnar venous blood samples were collected from all the participants three times (fasting, between 6:00 AM and 7:00 AM): before surgery (0), 1 day (1) and 4 days (2) after surgery. Baseline blood samples (0) were used for routine pre-operative laboratory assessment, including complete blood count, basic coagulation tests, C-reactive protein, glucose electrolyte levels and oxidative stress markers including TAS and TBARS measurements. Post-surgical blood samples (1 and 2) included assessment of TAS and TBARS only. Next, ratios expressing changes of these levels in time were calculated (1 : 0, 2 : 1; 2 : 0). The quotient of approximately 1.00 for any TAS ratio or any TBARS ratio means that there were no changes of total antioxidant status or lipid peroxidation products in plasma between the time points indicated. We also proposed an index which evaluates changes in TAS concentrations in relation to changes in TBARS levels, i.e. TAS (1 : 0)/TBARS (1 : 0). TAS were measured spectrophotometrically (RANDOX Laboratories, Crumlin, Antrim, UK) using Statfax 1904Plus (Awareness Technology, Palm City, FL) and TBARS were determined according to the Okhawa method [21 (link)] (Sigma reagents, Germany) using Specord M40 (Carl-Zeiss, Germany). The intra- and inter-assay coefficients of variation were as follows: TAS – 1.6% and 3.7%, TBARS – 1.9% and 3.8%.
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