Each SP and serum sample was divided into two aliquots in order to perform separate extractions of water- and fat-soluble antioxidants. The first aliquot of SP and serum (500 μL) was used for the extraction of hydrophilic antioxidants and biomarkers of oxidative/nitrosative stress, as described elsewhere [42 (link)]. Samples were briefly deproteinized by adding 1 mL of HPLC-grade CH3CN (VWR Chemicals, Briare, France) immediately centrifuged to pellet precipitated proteins and supernatants washed with HPLC-grade chloroform to remove organic solvent. The upper aqueous phases of serum and seminal plasma were diluted 3 and 10 times, respectively, with HPLC-grade water and then directly injected (100 μL) onto the HPLC Hypersil C-18, 250 × 4.6 mm, 5 μm particle size column (Thermo Fisher Scientific, Rodano, Milan, Italy), provided with its own guard column, for the analysis of ascorbic acid, GSH, uric acid, MDA, –NO2−, –NO3− and 8-OHdG.
The processing to extract fat-soluble antioxidants was carried out on the second aliquot of SP and serum using a method recently described in detail elsewhere [43 (link)]. SP or serum (500 µL) was added to 1 mL of HPLC-grade CH3CN. After vigorous vortexing for 60 s, these mixtures were incubated at 37 °C for 1 h in a water bath under agitation to allow the full extraction of lipid soluble compounds, and then centrifuged at 20,690× g for 15 min at 4 °C to precipitate proteins. Clear supernatants were directly used for the reversed phase HPLC analysis of all-trans-retinoic acid, all-trans-retinol, astaxanthin, lutein, zeaxanthin, trans-β-apo-8′-carotenal, γ-tocopherol, β-cryptoxanthin, α-tocopherol, lycopene, α-carotene, β-carotene and coenzyme Q10 using a Hypersil Gold RP C18, 150 × 4.6 mm, 5 µm particle size column, provided with its own guard column (Thermo Fisher Scientific, Rodano, Milan, Italy). All the aforementioned procedures were carried out by protecting samples from light in order to avoid the degradation of photo-sensitive molecules.
For both analyses, the HPLC was a Spectra System P4000 pump, equipped with a highly sensitive 5 cm light-path flow cell UV6000LP diode array detector, set up for acquisition between 200 and 550 nm wavelengths (Thermo Fisher Scientific, Rodano, Milan, Italy). Data acquisition and analysis were carried out using the ChromQuest software provided by the HPLC manufacturer.
Identification and quantification of the different compounds in chromatographic runs of SP and serum samples were performed by comparing retention times and absorption spectra of the various peaks with those of runs of ultrapure standard mixtures with known concentrations. In the final calculations, the sum of the concentrations of astaxanthin, lutein, zeaxanthin, trans-β-apo-8′-carotenal, β-cryptoxanthin, lycopene, α-carotene and β-carotene was performed, and the fat-soluble antioxidants were reported hereinafter as total carotenoids.
The processing to extract fat-soluble antioxidants was carried out on the second aliquot of SP and serum using a method recently described in detail elsewhere [43 (link)]. SP or serum (500 µL) was added to 1 mL of HPLC-grade CH3CN. After vigorous vortexing for 60 s, these mixtures were incubated at 37 °C for 1 h in a water bath under agitation to allow the full extraction of lipid soluble compounds, and then centrifuged at 20,690× g for 15 min at 4 °C to precipitate proteins. Clear supernatants were directly used for the reversed phase HPLC analysis of all-trans-retinoic acid, all-trans-retinol, astaxanthin, lutein, zeaxanthin, trans-β-apo-8′-carotenal, γ-tocopherol, β-cryptoxanthin, α-tocopherol, lycopene, α-carotene, β-carotene and coenzyme Q10 using a Hypersil Gold RP C18, 150 × 4.6 mm, 5 µm particle size column, provided with its own guard column (Thermo Fisher Scientific, Rodano, Milan, Italy). All the aforementioned procedures were carried out by protecting samples from light in order to avoid the degradation of photo-sensitive molecules.
For both analyses, the HPLC was a Spectra System P4000 pump, equipped with a highly sensitive 5 cm light-path flow cell UV6000LP diode array detector, set up for acquisition between 200 and 550 nm wavelengths (Thermo Fisher Scientific, Rodano, Milan, Italy). Data acquisition and analysis were carried out using the ChromQuest software provided by the HPLC manufacturer.
Identification and quantification of the different compounds in chromatographic runs of SP and serum samples were performed by comparing retention times and absorption spectra of the various peaks with those of runs of ultrapure standard mixtures with known concentrations. In the final calculations, the sum of the concentrations of astaxanthin, lutein, zeaxanthin, trans-β-apo-8′-carotenal, β-cryptoxanthin, lycopene, α-carotene and β-carotene was performed, and the fat-soluble antioxidants were reported hereinafter as total carotenoids.
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