All strains were grown at 20°C using standard C. elegans methods as previously described (Koh et al., 2018 (link)). Nematode growth media (NGM) agar plates were seeded with Escherichia coli strain OP50 for normal growth. C. elegans strains wild type N2, fat-1(bx24), mev-1(tk22), gst-4p::GFP::NLS(cl2166), sod-3p::GFP(cf1553) and bacteria strains OP50 were gifted from the Caenorhaditis Genetics Center. Trilinolenin [TG(54:9)] was obtained from Nu-Chek Prep and deuterated at bis-allylic position as previously described (Smarun et al., 2017 (link)) to obtained a mixture of 76% deuterated TG(54:9) [D-TG(54:9)]. Lipids were stored into an atmosphere of argon to prevent lipid oxidation. Lipids were freshly dissolved in PBS buffer (137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, pH 7.4) containing 0.1% Triton X-100 or kept at -80°C for later use prior to supplementing NGM agar plate as previously described (Deline et al., 2013 (link)). Butylated hydroxytoluene (BHT) and paraquat (PQ) were obtained from Sigma and Acros Organics, respectively.
Butylated hydroxytoluene bht
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Spain, Australia, Italy, China, Canada, Poland
Butylated hydroxytoluene (BHT) is a synthetic phenolic antioxidant used as a preservative in laboratory environments. It functions by inhibiting oxidation processes, thereby preventing degradation of sensitive materials.
Automatically generated - may contain errors
Lab products found in correlation
Gallic acid, by Merck Group (18 mentions)
Ascorbic acid, by Merck Group (16 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (16 mentions)
Quercetin, by Merck Group (13 mentions)
Methanol, by Merck Group (11 mentions)
Methanol, by Thermo Fisher Scientific (10 mentions)
Rutin, by Merck Group (9 mentions)
β carotene, by Merck Group (8 mentions)
Chloroform, by Merck Group (8 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (8 mentions)
Trichloroacetic acid, by Merck Group (7 mentions)
Caffeic acid, by Merck Group (6 mentions)
Chloroform, by Thermo Fisher Scientific (5 mentions)
Tween 80, by Merck Group (5 mentions)
Acetonitrile, by Merck Group (5 mentions)
Hplc grade methanol, by Thermo Fisher Scientific (5 mentions)
Ammonium acetate, by Merck Group (5 mentions)
Ethanol, by Merck Group (5 mentions)
Sodium hydroxide (naoh), by Merck Group (5 mentions)
N hexane, by Merck Group (5 mentions)
136 protocols using butylated hydroxytoluene bht
1
C. elegans Growth and Lipid Supplementation
2
Analytical Quantification of Metabolites
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All reagents and chemicals were of analytical grade and applied without further purification. HPLC-grade deionized water was used during all experiments.
α-KG, 5-HMF, NASeLM, NALM, and glucose were supplied by CYL-Pharma (Lassnitzhöhe, Austria). NaCl, KCl, KH2PO4, MgSO4, CaCl2, NaHCO3, and dextrose were obtained from Roth (Karlsruhe, Germany). Acetonitrile, ammonium acetate, 1-butanol, ethanol, HPLC-grade water, hydrogen peroxide, and HCl were obtained from Merck (Darmstadt, Germany). Albumin from human serum, angiotensin 1-7 acetate salt hydrate, butylated hydroxytoluene (BHT), 2,4-dinitrophenylhydrazine (DNPH), ethyl acetate, guanidine-hydrochloride, malondialdehyde tetra-butyl-ammonium salt (MDA), 2-thiobarbituric acid (TBA), trichloroacetic acid (TCA), and tris(hydroxymethyl)aminomethane (Tris) were obtained from Sigma-Aldrich (Vienna, Austria).
α-KG, 5-HMF, NASeLM, NALM, and glucose were supplied by CYL-Pharma (Lassnitzhöhe, Austria). NaCl, KCl, KH2PO4, MgSO4, CaCl2, NaHCO3, and dextrose were obtained from Roth (Karlsruhe, Germany). Acetonitrile, ammonium acetate, 1-butanol, ethanol, HPLC-grade water, hydrogen peroxide, and HCl were obtained from Merck (Darmstadt, Germany). Albumin from human serum, angiotensin 1-7 acetate salt hydrate, butylated hydroxytoluene (BHT), 2,4-dinitrophenylhydrazine (DNPH), ethyl acetate, guanidine-hydrochloride, malondialdehyde tetra-butyl-ammonium salt (MDA), 2-thiobarbituric acid (TBA), trichloroacetic acid (TCA), and tris(hydroxymethyl)aminomethane (Tris) were obtained from Sigma-Aldrich (Vienna, Austria).
3
HPLC-based Metabolite Profiling
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High-performance liquid chromatography (HPLC)-grade methanol, chloroform, and water were purchased from Fisher Scientific (Pittsburg, PA). HPLC-grade hexane was purchased from Honeywell Burdick & Jackson (Muskegon, MI). Butylated hydroxytoluene (BHT), myristic-d27 acid, methoxylamine hydrochloride, pyridine, and ammonium acetate were purchased from Sigma-Aldrich (St. Louis, MO). BSTFA [N,O-bis(trimethylsilyl) trifluoroacetamide] containing 1% trimethylchlorosilane (TMCS) was purchased from Alfa Aesar (Ward Hill, MA).
4
Salmon Bone Oil Enzymatic Extraction
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Salmon frame bone oil (SFBO) was collected from Seawell Co., Ltd. (frozen salmon was imported from Norway), Haeundaegu, Busan, Republic of Korea. Lipase Novozymes-435 (Candida antarctica) and Lipozyme RMIM (Rhizopus miehei) immobilized in macroporous anion exchange resin was bought from Novozymes A/S, Denmark. Fatty acid methyl esters (FAME), astaxanthin standard, p-anisidine, DPPH, ABTS, ascorbic acid, trolox, and butylated hydroxytoluene (BHT) were obtained from Sigma–Aldrich Co., St. Louis, Missouri, USA. Urea purity >99.00% was purchased from Junsei Chemical Co., Ltd., Tokyo, Japan. Analytical or HPLC graded solvents only were used in this study.
5
Optimization of β-CD Fungicide Extraction
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β-CD pharma grade (MW = 1,134.98 g mol−1, 98.5% purity) was obtained from Cyclolab, Ltd. (Budapest, Hungary). AITC (MW = 99.15 g mol−1) with 95% purity, and butylated hydroxytoluene (BHT) as a stabilizer (<0.1%) was purchased in Sigma Aldrich (Santiago, Chile). Acetonitrile with HPLC grade and technical grade solvents were acquired from Merck S.A. (Santiago, Chile). Botrytis cinerea (B05.10 strain) was gently provided by Center of Plant Biotechnology, Faculty of Biological Science, Universidad Andres Bello, Chile.
6
Synthesis and Characterization of Antioxidants
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Methanol was obtained from BDH chemicals limited, Poole-England. Caffeic acid (CA) and 2,3-dihydroxybenzoic acid (DHBA) were obtained from Sigma, Switzerland. Desferroxamine B (FOB) was obtained from Ciba-Geigy Ltd. (now Norvatis), Switzerland. Benzohydroxamic acid (BHA) was obtained from Sigma, United Kingdom. Butylated Hydroxy toluene (BHT), Ferrozine and DPPH were obtained from Sigma-Aldrich Inc., UK.
7
DPPH Radical Scavenging Activity of Cartilage Sulfate
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A previously described standard procedure was used for the measurement of 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity [21] (link). Briefly, 1 mL of DPPH (100 μM, Sigma–Aldrich, USA) in ethanol and 1 mL of antler CS fraction at different concentrations of CS (0.625–10 mg/mL on) in 100 mM Tris–HCl buffer (pH 7.4) were mixed. This reaction mixture was shaken and incubated for 20 min in the dark at room temperature. The absorbance was measured at 517 nm against a blank control (100 mM Tris–HCl buffer). Measurements were performed in triplicate over a 60-s period for each sample. The DPPH radical scavenging activity, namely the inhibitory ratio, was calculated using the following equation: scavenging activity (%) = (1 − Asample/Ablank) × 100, where Ablank is the absorbance of the blank. Lower absorbance of the reaction mixture indicates higher free radical scavenging activity. Ascorbic acid and butylated hydroxytoluene (BHT) (Sigma–Aldrich, USA) were used as positive controls. Two CS from bovine cartilage (C6737, Sigma–Aldrich, USA) and shark cartilage (C4384, Sigma–Aldrich, USA) were used as reference CS. The results were presented as the means of experiments performed in triplicate ± standard deviation.
8
Isolation and Analysis of Dolichols
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For the isolation and analysis of dolichols, HPLC or p.a. grade organic solvents from POCh were used (Gliwice, Poland), chromatographic columns and TLC plates were from Merck (Darmstadt, Germany), and other chemicals were of p.a. quality and were purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany).
For determination of phospholipids, the following standards from Avanti Polar Lipids (Alabaster, AL, USA) were used: 1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (14:0/14:0 phosphatidylglycerol (PG)); 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (12:0/12:0 phosphatidylethanolamine (PE)); 1,2-dimyristoyl-sn-glycero-3-phosphocholine (14:0/14:0 phosphatidylcholine (PC)); 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-myo-inositol) ammonium salt (16:0 phosphatidylinositol (PI)); 1,2-dimyristoyl-sn-glycero-3-phospho-l-serine sodium salt (14:0/14:0 phosphatidylserine (PS)), 1′,3′-bis[1–dimyristoyl-sn-glycero-3-phospho]-sn-glycerol ammonium salt (14:0 cardiolipin) and 1,2-dimyristoyl-sn-glycero-3-phosphate sodium salt (14:0/14:0 phosphatidic acid (PA)). Standards of polyisoprenoids were from the Collection of Polyprenols, Institute of Biochemistry and Biophysics, PAS, (IBB PAS) Warsaw. Butylated hydroxytoluene (BHT) was from Sigma-Aldrich. All other chemicals were from Avantor Performance Materials (Gliwice, Poland).
For determination of phospholipids, the following standards from Avanti Polar Lipids (Alabaster, AL, USA) were used: 1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (14:0/14:0 phosphatidylglycerol (PG)); 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (12:0/12:0 phosphatidylethanolamine (PE)); 1,2-dimyristoyl-sn-glycero-3-phosphocholine (14:0/14:0 phosphatidylcholine (PC)); 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-myo-inositol) ammonium salt (16:0 phosphatidylinositol (PI)); 1,2-dimyristoyl-sn-glycero-3-phospho-l-serine sodium salt (14:0/14:0 phosphatidylserine (PS)), 1′,3′-bis[1–dimyristoyl-sn-glycero-3-phospho]-sn-glycerol ammonium salt (14:0 cardiolipin) and 1,2-dimyristoyl-sn-glycero-3-phosphate sodium salt (14:0/14:0 phosphatidic acid (PA)). Standards of polyisoprenoids were from the Collection of Polyprenols, Institute of Biochemistry and Biophysics, PAS, (IBB PAS) Warsaw. Butylated hydroxytoluene (BHT) was from Sigma-Aldrich. All other chemicals were from Avantor Performance Materials (Gliwice, Poland).
9
Multimodal Analysis of Bioactive Compounds
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Acetonitrile, methanol water and dichloromethane (Merck; Darmstadt, Germany) were used for liquid chromatography diode array detection (LC-DAD) analysis and liquid chromatography tandem mass spectrometry (LC-MS/MS). Ethanol absolute and chloroform were obtained from VWR Chemicals (Radnor, PA). Hexane, butylated hydroxytoluene (BHT), formic acid (99% for mass spectrometry) along with analytical standards (chicoric acid, chlorogenic acid, lutein, β-carotene, violaxanthin, neoxanthin, β-cryptoxanthin, and cyanidin) were purchased from Sigma-Aldrich (St. Louis, MO). Ultrapure water was obtained from a Milli-Q Gradient A10 water purification system.
10
Lipid Extraction and FAME Analysis
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Solvents and chemicals used for lipid extraction and FAME preparation were of analytical grade. Chloroform, dichloromethane, methanol, n-hexane, ethanol, hydrochloric acid, ammonia, anhydrous sodium sulfate, butylated hydroxytoluene (BHT), potassium chloride, sodium chloride, potassium hydroxide, sodium hydroxide, and hexadecane (used as internal standards) were purchased from Sigma Aldrich (Darmstadt, Germany). The standard mix of 37 FAMEs and the standard mix of C4:0–C24:0 saturated FAMEs were purchased from Supelco (Bellefonte, PA, USA).
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