3 5 di tert 4 butylhydroxytoluene

Whole frozen lungs and spleens were pulverized using a CP02 automated cryoPREP® (Covaris). Briefly, the frozen tissue was transferred into a tissue tube (Covaris, extra thick) and cooled down by dipping in liquid nitrogen for 60 s. Next, the sample was pulverized with an impact level of four. This step was repeated. 50 mg of powder were immediately extracted with 500 µl ice cold methanol (Roth®) containing 0.1% 3,5-Di-tert-4-butylhydroxytoluene (Sigma-Aldrich) and 500 µl ice cold water. Next, 100 µl internal standard consisting of 12-HETE-d₈ and 13-HODE-d₄ (both 100 ng/ml in acetonitrile; Cayman chemicals) was added. Alkaline hydrolysis was performed by adding 300 µl of sodium hydroxide (10 mol/l; Sigma-Aldrich) followed by an incubation for 30 min at 60 °C. Immediately after hydrolysis, the pH was adjusted to a value of 6 using acetic acid (10 mol/l, VWR). For EDTA plasma samples, an aliquot of 100 µl was hydrolyzed and extracted using 500 µl ice cold methanol with 0.1% 3,5-Di-tert-4-butylhydroxytoluene, 500 µl ice cold water, and 100 µl internal standard solution. Samples were hydrolyzed with sodium hydroxide as described for the tissue material. Afterwards, solid phase extraction was done for all samples types as previously described (Schultz et al., 2019 ).

LC-MS grade solvents and reagents as water, methanol, ammonium formate, formic acid, and acetate acid, as well as 3,5-Di-tert-4-butylhydroxytoluene (BHT), were from Sigma-Aldrich (Saint Louis, MO, USA). Ethanol and HPLC analytical grade chloroform were, respectively, from J.T. Baker (Center Valley, PA, USA) and Carlo Erba (Cornaredo, MI, Italy). Potassium hydroxide was from Merk Millipore (Burlington, MA, USA). Sphingosine (d17:1), sphinganine-1-phosphate (d17:1), ceramide (d18:1/12:0), sphingomyelin (d18:1/12:0) and glucosyl (β)ceramide (d18:1/12:0) were from Avanti Polar Lipids (Alabaster, AL, USA).

All reagents used were of analytical grade. Sodium pyruvate, dimethyl thiourea (DMTU), nordihydroguaiaretic acid (NDGA), ascorbic acid, histidine, xylenol orange (FOX), 2,2-diphenyl-1-picrylhydrazyl (DPPH), dimethylsulfoxide (DMSO), N,N-dimethyl-4-nitrosoaniline (DMNA), catalase, xanthine, xanthine oxidase, nitroblue tetrazolium (NBT), dL-penicillamine 2-thiobarbituric acid (TBA), α-tocopherol, Folin and Ciocalteu's phenol reagent, 3,5-di-tert-4-butylhydroxytoluene (BHT), and 5-fluoro-2′-deoxyuridine were purchased from Sigma-Aldrich (Toluca, Mexico, or Sigma, St. Louis, MO). Absolute ethanol, hydrogen peroxide (H₂O₂), sulfuric acid (H₂SO₄), methanol, ethylenediaminetetraacetic acid disodium salt (EDTA), NaCl, KH₂PO₄, NaHPO₄·7H₂O, NaOH, and H₂SO₄ were purchased from Merck-Mexico. Sodium hypochlorite (NaOCl) was purchased from Hycel (Mexico City, Mexico).

Methanol, LC-MS grade water, 3,5-Di-tert-4-butylhydroxytoluene (BHT), and ammonium formate were from Sigma-Aldrich (Saint Louis, MO, USA). Ethanol and HPLC analytical grade chloroform were, respectively, from J.T. Baker (Center Valley, PA, USA) and Carlo Erba (Cornaredo, MI, Italy). Potassium hydroxide was from Merk Millipore (Burlington, MA, USA). Acetic and formic acid were from Fluka-Analitical (Honeywell, Morris Plains, NJ, USA).
Sphinganine (d17:0), sphinganine-1-phosphate (d17:0), C12 Ceramide, Sphingomyelin (d18:1/12:0), and Glucosyl (β) C12 Ceramide were from were from Avanti Polar Lipids (Alabaster, AL, USA).

glycerol monomethacrylate (GMA, purity 97%) was obtained from GEO speciality chemicals (Hythe, UK) and was used as received. 2,2,2-Trifluoroethylmethacrylate (TFEMA, 99%), lauryl methacrylate (LMA, 96%), n-dodecane (>99%), glycerol (>99%), sucrose (>99.5%), Nile red, CD₃OD, tetrahydrofuran (THF), dimethylformamide (DMF) (CD₃)₂CO, lithium bromide (LiBr), CDCl₃, dimethyl sulfoxide (DMSO), triethylamine, 3,5-di-tert-4-butylhydroxytoluene (BHT), toluene, benzyl methacrylate (BzMA, 96%), 4,4′-azobis(4-cyanovaleric acid) (ACVA, >97%), benzophenone (>99%), pyrene (>99%), 2-cyanopropyldithiobenzoate (CPDB, >97%), 2-phenylethanethiol, sodium hydride (60% in mineral oil), diethyl ether, carbon disulfide, iodine, sodium thiosulfate, sodium sulfate, ethyl acetate and n-hexane were purchased from Sigma Aldrich (UK). tert-Butyl peroxy-2-ethylhexanoate (Trigonox 21S or T21s) initiator was supplied by AkzoNobel (The Netherlands) and sodium dodecylsulfate (SDS) was obtained from BDH laboratory supplies (Poole, UK). benzyl methacrylate was passed through basic alumina prior to use; all remaining reagents were used as received unless otherwise stated. Deionized water (pH 6.1 at 20 °C) was used for all experiments described herein. All solvents used were of HPLC grade.

Chloroform (Carlo Erba Reactifs SDS, Val-de-Reuil, France), acetonitrile, methanol, isopropanol, water of LC-MS grade (J.T. Baker, Phillipsburg, NJ, USA) and 3,5-di-tert-4-butylhydroxytoluene (Sigma–Aldrich, Saint-Quentin Fallavier, France) were used to perform cell lipid extraction and to prepare mobile phase for liquid chromatography. All commercial lipid standards were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA) and are listed in Table S1 of the Supplementary Materials.

Alpha-amylase (EC 3.2.1.1)—77 U/mg from human saliva type XIII-A, rutine hydrate, quercetin, kaempferol, trans-resveratrol, DPPH (2,2-Diphenyl-1-picrylhydrazyl), trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid), neocuproine, ammonium acetate, Copper (II) chloride dehydrate, 2,4,6-tris (2-pyridyl)-s-triazine (TPTZ), iron(III) chloride hexahydrate and 3,5-di-tert-4-butylhydroxytoluene (BHT) were purchased from Sigma-Aldrich Chemie (Schnelldorf, Germany). Delphinidin-3-O-galactoside chloride for HPLC, Cat. No. 0905, delphinidin-3-O-glucoside chloride for HPLC, Cat. No. 0938 S, malvidin-3-O-glucoside chloride for HPLC, Cat. No. 0911 S, cyanidin-3-O-glucoside chloride for HPLC and Cat. No. 0915 S were purchased from Extrasynthese (Lyon, France). HPLC grade methanol, acetonitrile and formic acid were obtained from Merck (Darmstadt, Germany). Starch soluble GR (500 gm), 3,5-dinitrosalicylic acid, 2-[4-(2-Hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid buffer substance HEPES, Folin–Ciocalteu’s phenol reagent, formic acid, gallic acid, potassium chloride and sodium acetate were purchased from Merck (Darmstadt, Germany).