Curcuminoids and their metabolites from fermented fecal slurries were extracted following the method reported by Tan et al. [11 (link)], with slight modifications. Briefly, these compounds were extracted by adding 1 mL of ethyl acetate, vortexed for 1 min, and sonicated for 10 min in an ultrasonic bath. The upper organic layer (900 µL) was transferred to a clean microfuge tube. The above extraction procedure was repeated once using just the lower layer and transferring 1000 µL in a clean microfuge tube. Both upper layers were dried for about 2 h at room temperature through a centrifugal vacuum concentrator (SpeedVac Savant SPD121P, Thermo Fisher Scientific Inc., San Jose, CA, USA). The residue was then reconstituted in 200 µL of aqueous acetonitrile, acidified with formic acid (60:39.8:0.2, v/v), vortexed, sonicated for 5 min and centrifuged at 14,462× g for 5 min before uHPLC-MSn analyses.
Speedvac savant spd121p
Manufactured by Thermo Fisher Scientific
Sourced in United States
The SpeedVac Savant SPD121P is a benchtop centrifugal evaporator designed for sample concentration and solvent removal. It features a compact design and accommodates a variety of sample holders for efficient drying of samples.
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5 protocols using speedvac savant spd121p
1
Extraction and Analysis of Curcuminoids
2
Extraction and Analysis of Quercetin Metabolites
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Fecal metabolites produced during the in vitro fecal incubation of unformulated and phytosome-formulated quercetin were extracted adopting the method reported by Bresciani et al. [18 (link)], with slight modifications. Briefly, 300 µL of each fermented sample was extracted with 0.1% (v/v) formic acid in ethyl acetate, vortexed for 30 s, sonicated for 10 min in an ultrasonic bath, vortexed for 30 s, and re-sonicated for 5 min. Finally, samples were centrifuged at 14,460× g for 10 min and the upper organic layer was transferred to a clean microfuge tube. After the first extraction, the residual pellet of the fermented samples was re-extracted following the same procedure, using 500 μL of the same solvent. Finally, supernatants were pooled and brought to dryness for about 2 h at room temperature through a centrifugal concentrator (SpeedVac Savant SPD121P, Thermo Fisher Scientific Inc., San Jose, CA, USA). Both dried residues were reconstituted in 50% (v/v) aqueous methanol acidified with 0.1% (v/v) formic acid (dilution factors of 1:10 and 1:2 for the analyses of native quercetin and its fecal metabolites, respectively), vortexed, and centrifuged at 14,460× g for 10 min before uHPLC-MSn analyses.
3
Purification and Characterization of KTCf20
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Wickerhamomyces anomalus Cf20 was inoculated at 1×10 6 cells/ml in YPD broth pH 3.0 and incubated at 25 ºC during 96 h. The culture was centrifuged at 10 000 ×g for 10 min and sterilized by filtration with 0.22 µm PVDF filters (Millipore). Glycerol was added to the cell-free supernatant to a final concentration of 10%.
For purification, 1 liter of crude extract was concentrated by ultrafiltration (Amicon YM30, 50 kDa, Millipore) to a final volume of 5 ml. The concentrated fraction was purified by size exclusion chromatography (SEC) through a 10×300 mm Sephadex G-75 column with 0.1 M citric acid/dibasic sodium phosphate pH 3.0 (BCF) at a flow of 0.9 ml/min. Active fractions were pooled and concentrated 10× at 30 ºC for 7 h in a SpeedVac Savant SPD 121P (Thermo Scientific, Thermo Electron Corporation, Ohio, USA). For experiments, dilutions of the concentrated KT were prepared in BCF pH 3.0.
Protein concentration was quantified by the Bradford method.
Purified KTCf20 was subsequently analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using silver staining technique (Chevallet et al., 2006) .
In native electrophoresis, samples were analyzed in a 5% continuous acid polyacrylamide gel soaked in a 0.375 M acetic acid-KOH solution, pH 4.0 (Bollag and Edelstein, 1991) .
For purification, 1 liter of crude extract was concentrated by ultrafiltration (Amicon YM30, 50 kDa, Millipore) to a final volume of 5 ml. The concentrated fraction was purified by size exclusion chromatography (SEC) through a 10×300 mm Sephadex G-75 column with 0.1 M citric acid/dibasic sodium phosphate pH 3.0 (BCF) at a flow of 0.9 ml/min. Active fractions were pooled and concentrated 10× at 30 ºC for 7 h in a SpeedVac Savant SPD 121P (Thermo Scientific, Thermo Electron Corporation, Ohio, USA). For experiments, dilutions of the concentrated KT were prepared in BCF pH 3.0.
Protein concentration was quantified by the Bradford method.
Purified KTCf20 was subsequently analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using silver staining technique (Chevallet et al., 2006) .
In native electrophoresis, samples were analyzed in a 5% continuous acid polyacrylamide gel soaked in a 0.375 M acetic acid-KOH solution, pH 4.0 (Bollag and Edelstein, 1991) .
4
Modulation of Cholesterol Homeostasis in Hep3B Cells
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After plating for 24 h, Hep3B cells were washed in PBS without Ca2+/ and Mg2+ and treated with DMEM supplemented with 10% (v/v) lipoprotein-deficient foetal bovine serum (Thermofisher, Waltham, MA, USA) plus the relative treatment. Treatments were as follows: 5 µM simvastatin (Sigma Aldrich, St. Louis, MO, USA) dissolved in dimethyl sulfoxide (DMSO) (Sigma Aldrich, St. Louis, MO, USA) plus 10 µM mevalonate (Sigma Aldrich, St. Louis, MO, USA) dissolved in ethanol (Thermofisher, Waltham, MA, USA) (statin treatment), 20 mg/mL methyl-ß-cyclodextrin plus vehicle (0.1% DMSO and 0.02% ethanol) (cyclodextrin treatment) and 20 mg/mL cholesterol-loaded methyl-ß-cyclodextrin plus vehicle (0.1% DMSO and 0.02% ethanol) (COH-cyclodextrin treatment) or vehicle alone (DMSO and ethanol) (control). methyl-ß-cyclodextrin and cholesterol-loaded methyl-ß-cyclodextrin (Sigma Aldrich, St. Louis, MO, USA) were prepared as follows: 5% methyl-ß-cyclodextrin (w/v) in H2O +/− 15 mg/mL cholesterol in 100% ethanol (methyl-ß-cyclodextrin: cholesterol at 10:1) was stirred for 30 min at 80 °C on a heat block before being dried down using a Savant SPD121P SpeedVac (Thermofisher, Waltham, MA, USA). Prior to experiments, treatments were freshly dissolved in molecular grade water.
5
DBS Metabolites Extraction and Quantification
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The complete DBS sample processing flowchart is shown in Supplementary Figure S-3 . Based on material availability, one or two 3-mm DBS punches (equivalent to 3μl and 6μl of whole blood, respectively) were reconstituted in 150μl of 50mM ammonium bicarbonate buffer in an orbital shaker (1,600rpm, 60min). We removed a volume of 5μl for BCA (section Protein Extraction and Processing Protocol, and Mass Spectrometry Assays) and dried the remaining sample in a vacuum concentrator centrifuge (Savant SPD121 P SpeedVac, Thermo Fisher). A volume of 400μl of 80% isopropanol (v/v) was added to a dry sample and vortexed at 200rpm for 20min. The sample was briefly centrifuged, supernatant quantitatively transferred into a 96-well plate, and dried in the SpeedVac. Dry extracts were redissolved in 10μl 5% isopropanol (v/v) containing isotopically labeled standards: 200nM [13C6] indole-3-acetate, 2000nM [2H5] L-kynurenine, 20,000nM [13C11][15N2] L-tryptophan, 50nM [2H5][15N2] indole-3-acetamide, 200nM [13C6] anthranilate (Supplementary Table S-4 ). Several solvents, i.e., 80% isopropanol, 100% isopropanol, 50% isopropanol, 80% acetonitrile, and 100% acetonitrile, were tested for optimal extraction recoveries of metabolites from DBS (n=3). The optimal extraction solvent was 80% isopropanol (data not shown).
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