The concentration of vitamin E in the plasma samples was quantified as described elsewhere [32 (link)]. Vitamin E (α-tocopherol) was extracted directly without saponification. Duplicate plasma aliquots were mixed with a dibasic sodium phosphate buffer (0.054 M) adjusted to pH 7.0. Tocopherol was extracted via centrifugation (600× g for 10 min at 4 °C) after the addition of hexane to the mixture. After the evaporation of the upper layer, the remaining residue was dissolved in ethanol. Tocopherol was analysed using reverse-phase HPLC (HP 1100, equipped with a diode array detector) (Agilent Technologies, Waldbronn, Germany) [32 (link)]. Identification was carried out using the pure compound (Sigma-Aldrich, Alcobendas, Madrid) and quantification (µg of α-tocopherol per mL of plasma) was carried out by means of a standard curve built with the pure compound.
Pure compound
Manufactured by Merck Group
Sourced in Spain
Pure compounds are high-purity chemical substances designed for use in various laboratory applications. They provide a consistent and well-defined composition to support accurate experimentation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Hp1100, by Agilent Technologies (1 mentions)
Sodium acetate, by Merck Group (1 mentions)
Folin ciocalteu phenol reagent, by Merck Group (1 mentions)
Chromasolv, by Merck Group (1 mentions)
Ethyl acetate hplc grade, by Merck Group (1 mentions)
Agilent gc ms 5975 7890, by Agilent Technologies (1 mentions)
Victor 3v 1420 multilabel plate reader, by PerkinElmer (1 mentions)
Dmem glutamax, by Thermo Fisher Scientific (1 mentions)
Sh sy5y, by Thermo Fisher Scientific (1 mentions)
Activated charcoal, by Merck Group (1 mentions)
Celltiter glo luminescent cell viability assay, by Promega (1 mentions)
Hp 1200, by Agilent Technologies (1 mentions)
6 protocols using pure compound
1
Quantification of Plasma Vitamin E Levels
2
Spectrophotometric Determination Protocols
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The reagents used for spectrophotometric determinations (sodium acetate, aluminum chloride, Folin–Ciocalteu phenol reagent, reagent-grade sodium carbonate, sodium acetate buffer, potassium chloride buffer) and solvents were bought from Merck (Darmstadt, Germany)
All standards and solvents were of HPLC (Chromasolv) quality and were purchased either from Sigma-Aldrich or from Merck (Darmstadt, Germany). The pure compounds used as standards were obtained from Sigma-Aldrich (Darmstadt, Germany) and the purity grade was at least 95%.
All standards and solvents were of HPLC (Chromasolv) quality and were purchased either from Sigma-Aldrich or from Merck (Darmstadt, Germany). The pure compounds used as standards were obtained from Sigma-Aldrich (Darmstadt, Germany) and the purity grade was at least 95%.
3
Pentadecane and Succinic Acid Analysis
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Following induction and incubation, cell cultures were centrifuged, and the supernatant decanted. In a typical pentadecane assay, 3 ml of supernatant vigorously mixed with 1 ml of 1M NaCl and 1 ml pure high-performance liquid chromatography (HPLC) grade ethyl acetate (Sigma Aldrich). After 1 h of shaking, the ethyl acetate extract was separated by centrifugation (10 min at 4000 g) and obtaining the top layer. Extracts were analyzed by gas chromatography–mass spectrometry (GC-MS) on an Agilent GC-MS 5975/7890 (Agilent Technologies) with the HP-5MS column (30 m length, 0.50 mm diameter). Following a 1–3 μl split-less injection, the inlet temperature was held at 100°C for 3 min and ramped up to 320°C at 30°C per minute. For quantification, standard curves of pentadecane and hexadecanol were generated, by using the pure compounds (Sigma Aldrich). Succinic acid production assays were similarly performed on decanted supernatant. Roche (r-biopharm) enzymatic succinic acid assay kits (CN: 10176281035) were used to measure production levels using a Victor3V 1420 Multilabel plate reader (Perkin Elmer).
4
Cell Line Toxicity Screening Protocol
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All chemical solutions were prepared by dissolving pure compounds (Sigma Aldrich St Louis MI USA) in dimethylsulfoxide (DMSO). Cells were cultivated in GlutaMAX Dulbecco modified Eagle medium (GlutaMAX DMEM, Thermo Fisher Scientific) with 10% (HEK293) or 12% (C17.2 or SHSY5Y) of newborn calf serum (Thermo Fisher Scientific). Endogenous TH were depleted from the serum by stripping with activated charcoal (Sigma Aldrich, St Louis MI USA) to prevent background activation of reporter constructs. The toxicity of each chemicals was tested on each cell line using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison WI, USA). Toxic concentrations (>20% mortality after 24 hours) were used to define the maximum concentration usable in reporter assays, defined as 10% of the lowest toxic concentration.
5
Cheese Quality Analysis Protocol
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Grated cheese samples were analyzed in duplicate for salt content by a potentiometric method (IDF, 1981) , fat content of cheese was analyzed using the Röse-Gottlieb method (IDF, 1996) and moisture content by oven-drying at 102°C for 5 h (IDF, 1982) . The pH of cheese was assessed after 90, 180, and 270 d of ripening by blending 12 mL of H 2 O with 20 g of grated cheese and measuring using a standard pH meter (Mettler Toledo MP220; Mason Technology Ltd., Dublin, Ireland) according to the method of the British Standards Institution (1976) . Calcium content analysis was performed by inductively coupled plasma optical emission spectrometry following microwave-assisted acid digestion. Trans-β-carotene was saponified using ethanolic potassium hydroxide solution for 16 h at room temperature and extracted once with ethanol:hexane (4:3 vol/vol) and 2 times with hexane. Trans-β-carotene analysis was performed by reverse-phase (RP)-HPLC with an UV diode array detector (UV/DAD) at 452 nm. The calibration standards used were pure compounds (purity >98%) from Sigma Aldrich. The purity of the standard for each calibration was determined by a series of spectrophotometric measurements (UV at 340, 455, and 483 nm). Both methods were performed by Eurofins Food Testing (Dublin, Ireland). All chemical analysis was performed after 90 d of ripening.
6
Vitamin E Quantification in Sows and Piglets
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The Vitamin E (α-tocopherol) concentration in sows’ and piglets’ plasma was extracted by the direct procedure described by Rey et al. [21 (link)]. Briefly, dibasic sodium phosphate buffer (0.054 M) adjusted to pH 7.0 was added to duplicate plasma aliquots. Tocopherol was extracted by centrifugation (600× g during 10 min at 4 °C) after the addition of absolute ethanol and hexane. Then, the upper layer was collected and evaporated by N2 stream and the remaining residue was dissolved in ethanol and injected into an HPLC (HP 1200, equipped with a diode array detector and a reverse RP-18 column) (Agilent Technologies, Waldbronn, Germany) [21 (link)]. Identification and quantification were carried out using the pure compound (Sigma-Aldrich, Alcobendas, Madrid). Results were expressed as µg of α-tocopherol per mL of plasma.
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