Pyridine

The free metabolite pool of muscle samples was extracted as described previously with small modifications^{13 (link)}. Frozen tissue of rat TA (n = 7) was weighed and 20 mg of tissue per sample was solubilized in methanol-chloroform-water (MCW; 5:2:1) (Sigma-Aldrich, Germany), containing 2 µg/ml cinnamic acid (Sigma-Aldrich, Germany) as internal standard. After addition of MCW, all samples were shaken twice at 6000RPM for 1 min using a Precellys 24 (Bertin Technologies, France) to homogenize them. This step was repeated after 10 µl ddH2O * mg-1 of tissue was added to the muscle samples. For phase separation of polar and lipid intermediates, all samples were spun for 10 min at 20,000 g and 4 °C. The supernatant containing the polar phase was then collected and dried for 3 h in a SpeedVac (Thermo Scientific, Germany). For derivatization, the dried samples were dissolved in 20 µl methoxyamine hydrochloride solution (Sigma-Aldrich, Germany) (40 mg/ml in pyridine [Roth, Germany]) and incubated for 90 min at 30 °C in a Thermomixer (Eppendorf, Germany). Subsequently, 80 µl of N-methyl-N- (trimethylsilyl)trifluoroacetamide (MSTFA) (Machery-Nagel, Germany) containing a retention index standard was added to the samples and incubated at 37° for 45 min. The supernatant was then collected and transferred into appropriate vials for GC-MS measurement.

Formic acid, ammonium acetate, and 3-nitrophenylhydrazine (#N21804) were acquired from Sigma-Aldrich (St. Louis, MO), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, #2156.1) and pyridine (#CP07.1, 99%) were from Roth (Karlsruhe, Germany). Authentic acylcarnitine standards were obtained from Cambridge Isotope Laboratories (Andover, MA, #NSK-B), and Sigma-Aldrich (2-methylbutyryl-L-carnitine #50405, isovaleryl-L-carnitine #51371, valeryl-L-carnitine #04265, oleoyl-L-carnitine (C18:1) #19945, and O-succinyl-L-carnitine (C4-DC) #04609. All other reagents were of analytical LC-MS grades.

n-Propanol (for synthesis), squalene (99%) and a technical β-sitosterol product (55.5% β-sitosterol, 34.2% campesterol [16 (link)]) were purchased from Merck (Darmstadt, Germany). Silica gel 60 (for column chromatography), 3-phenyl propionic acid (99%), 5α-cholestane (97%), cholesterol (> 99%) and stearic acid (99%) were ordered from Sigma-Aldrich (Steinheim, Germany). Dichloromethane (99.8%), mEthanol (HPLC gradient grade) and n-hexane (HPLC gradient grade) were purchased from Th. Geyer (Renningen, Germany). Ethanol (distilled before use, 7.4% water content), sodium chloride (99%), pyridine (99%) and sulphuric acid (96%) were obtained from Carl Roth (Karlsruhe, Germany). Tripalmitin (95%, containing traces of dipalmitoylglycerol), cholesteryl stearate (97%) and elaidic acid (99%) were from Fluka (Taufkirchen, Germany). Palmitic acid (98%) was purchased from Riedel de Häen (Seelze, Germany). 8-Phenyl octanoic acid (97%) and n-hexanol (99%) were from Thermo Fisher Scientific (Kandel, Germany). The trimethylsilylating reagent SILYL-991 was ordered from Macherey–Nagel (Düren, Germany). Nitrogen and helium 5.0 quality originated from Westfalen company (Münster, Germany). Dihydrolanosterol [19 ] and α-tocopherol [23 (link)] were isolated autonomously with CCC. Demineralised water was obtained through the in-house supply (Hohenheim, Germany).

D-(+)-glucuronic acid γ-lactone, pyridine, methyl-(p-hydroxymethyl) benzoate, triethylamine (TEA), acetic acid, dichloromethane (DCM), acetonitrile (ACN), methanol (MeOH), heptane, ethyl acetate, toluene, tetrahydrofuran (THF), N, N-dimethylformamide (DMF) were ordered from Carl Roth. Tert-butyldimethylsilyl chloride (TBDMSCl), diphenyl phosphoryl azide (DPPA), lithium methoxide solution (LiOMe), 4-nitrophenyl chloroformate (Cl-COOPhNO₂) were ordered from Sigma. Doxorubicin hydrochloride salt (DOX·HCl) was ordered from Biomol.

All chemicals were used as received without further purification. Ni(NO₃)₂·6H₂O (97%), 2,6-H₂ndc (99%), dabco (99%), polyacrylic acid (PAA; M_w = 1,800), and dry methanol were purchased from Sigma Aldrich and N,N-dimethylformamide (DMF) of reagent grade purity from Fisher Chemical. Acetic acid (100%) and pyridine (99%), extra pure, were purchased from Carl Roth and Acros Organics, respectively.