β nicotinamide adenine dinucleotide 2 phosphate reduced tetrasodium salt hydrate β nadph

Fipronil (PESTANAL, analytical standard; product code: 46451; empirical formula: C₁₂H₄Cl₂F₆N₄OS; molecular weight: 437.15 g/mol; CAS number: 120068-37-3; ≥98.8% purity), quercetin (product code: 337951; molecular weight: 302.21 g/mol; CAS number: 849061-97-8; ≥95% purity), and curcumin [curcumin from Curcuma longa (Turmeric), powder; product code: C1386; molecular weight: 368.38 g/mol; CAS number. 458-37-7; assay ≥65% (HPLC)] were purchased from Sigma Aldrich (Germany) and used.
DL-Dithiothreitol, 5,5’-Dithiobis (2-nitrobenzoic acid) (DTNB), ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA), phenylmethylsulfonyl fluoride, potassium chloride, hydrogen peroxide solution, Triton X-100, Trizma base, pyrogallol, L-glutathion reduced, β-Nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate (β-NADPH), glutathion reductase, ammonium sulfate, trichloroacetic acid, 1-chloro-2,4-dinitrobenzene (CDNB), butylated hydroxytoluene, 2-thiobarbituric acid (TBA), bovine serum albumin, and disodium hydrogen phosphate were obtained from Sigma Aldrich. Hydrochloric acid, Folin–Ciocalteu’s phenol reagent, sodium hydroxide, and potassium dihydrogen phosphate were purchased from Merck (Germany).

RPMI-1640 medium was obtained from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). The antibiotic mixture penicillin/streptomycin (10.000 U/mL/10.000 mg/mL), heat-inactivated fetal bovine serum (FBS) and trypsin-EDTA (0.25%) were purchased from GIBCO Invitrogen (GIBCO, Barcelona, Spain). Hydrochloric acid (HCl), sodium hydrogen carbonate and Hank′s Balanced Salt Solution (HBSS) were obtained from Merck (Merck, Darmstadt, Germany). Deuterium oxide (D₂O) was provided by Eurisotop (Eurisotop, Saint-Aubin, France) and D₂O containing 0.05 wt% 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid (TSP) sodium salt was purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). Thiazolyl blue tetrazolium bromide (MTT), sodium pyruvate, β-nicotinamide adenine dinucleotide reduced disodium salt hydrate (β-NADH), β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate (β-NADPH), and dichlorodihydrofluorescein diacetate (DCFH-DA) were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). Solvents such as dimethyl sulfoxide (DMSO), methanol and chloroform were acquired from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). DMSO cell culture grade was obtained from PanReac (PanReac, Barcelona, Spain). Sunitinib malate (≥98%) and pazopanib (≥98%) were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA).

Sodium nitrate (NaNO₃), sodium nitrite (NaNO₂), 2,3-diaminonaphthalene (DAN, the derivatizing agent), nitrate reductase (from Aspergillus niger), β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate (β-NADPH), and flavin adenine dinucleotide disodium salt hydrate (FAD) were obtained from Sigma–Aldrich; ¹⁵N-NaNO₂ and ¹⁵N-NaNO₃ were purchased from Cambridge Isotope Laboratories.
Standard stock solutions of nitrite and nitrate used to establish calibration curves were individually prepared by dissolving NaNO₂ or NaNO₃ in deionized water to the desired concentrations. The linear range for NO₂⁻ was 0.03 to 2.0 μM, and each calibrator contained 0.5 nmol of the stable isotope-labeled internal standard (SIL-IS) ¹⁵N-NO₂⁻. The linear range for NO₃⁻ was 15.6 to 1000 μM, and each calibrator contained 6.25 nmol of the SIL-IS ¹⁵N-NO₃⁻. The calibrators were processed and analyzed as described later for the PE samples.