Glutathione reductase

The glutathione peroxidase (Gpx) activity was assayed in plasma, according to Lawrence and Burk [46 (link)] and liver and dorsal muscle as described by Fontagné-Dicharry et al. [21 (link)]. Gpx activity in plasma samples was evaluated immediately after thawing. In the case of liver and muscle, samples were rapidly thawed and homogenized in 10 volumes (w/v) of ice-cold saline for 3 min and centrifuged for 15 min at 4000× g and the supernatants collected to evaluate the activity of GPx. Gpx activity present in the supernatants was measured in a solution of 50 mM phosphate buffer (pH 7.4), 1 mM EDTA (Merck, Darmstadt, Germany), 2 mM sodium azide (Merck, Darmstadt, Germany), 2 mM reduced glutathione (GSH) (Merck, Darmstadt, Germany), 0.1 mM NADPH (Merck, Darmstadt, Germany), and 0.2 mM glutathione reductase (Merck, Darmstadt, Germany) following the reduction of H₂O₂ (50μM) at 30 °C and 340 nm. One unit of Gpx activity was reported as l mol NADPH consumed per min per mg of plasma protein, using the appropriate molar absorptivity coefficient for NADPH (6220 mol L⁻¹ cm⁻¹). Plasma proteins were measured by the method of Lowry et al. [47 (link)].

The Gpx activity was measured in plasma following the indications of Lawrence and Burk [79 (link)] and in the liver and in the dorsal muscle those of Fontagné-Dicharry et al. [80 (link)]. For plasma, Gpx activity was evaluated immediately after thawing. For liver and muscle, after thawing the samples on ice and homogenizing them in 10 volumes (w/v) of ice-cold saline for 3 min, they were centrifuged for 15 min at 4000× g before the activity of GPx was determined in the supernatants. GPx activity was measured in a solution of 50 mM phosphate buffer (pH 7.4), 1 mM EDTA (Merck, Darmstadt, Germany), 2 mM sodium azide (Merck, Darmstadt, Germany), 2 mM reduced glutathione (GSH) (Merck, Darmstadt, Germany), 0.1 mM NADPH (Merck, Darmstadt, Germany), and 0.2 mM glutathione reductase (Merck, Darmstadt, Germany). H₂O₂ (50 μM) reduction at 30 °C was measured at 340 nm in an Epoch spectrophotometer. One unit of Gpx activity was valued as 1 mol NADPH consumed per min per mg of plasmatic proteins, using the appropriate molar absorptivity coefficient for NADPH (6220 mol L⁻¹ cm⁻¹). Plasmatic protein measurement was performed following the method of Lowry et al. [81 (link)].

Buthionine sulfoximine (BSO), L-glutathione reduced, Glutathione reductase, 5-5′-dithiobis (2-nitrobenzoic acid) (DTNB), β-Nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate (NADPH), Triton-X and sulfosalicylic acid were from Sigma Aldrich.

Kits for Glucose, insulin, total cholesterol, triglycerides, HDLc and LDLc were purchased from Abbott Laboratories (Abbott Park, IL, USA). HsCRP kits were supplied by Beckman Corp (Brea, A, USA). The HbA1c kit was purchased from Menarini Diagnostics (Florence, Italy). The MPO kit was purchased from Cayman Chemical (Michigan, USA).
Glucose, trypan blue, arginine, glutathione reductase, H₂O₂, haemoglobin, RPMI1640 supplemented with 20 mM HEPES, HBSS, TNF-α, human serum albumin (HSA, Albuminate 25%) and fibronectin were obtained from Sigma-Aldrich (Sigma Chem. Co., St. Louis, MO, USA). Dextran was acquired from Fluka (St. Louis, MO, USA). HBSS was supplied by Cambrex (Verviers, Belgium). DCFH-DA and Mitosox tracker were provided by Calbiochem (San Diego, CA, USA). Dulbecco’s PBS—with (DPBS⁺) or without (DPBS^-) Ca²⁺ and Mg²⁺—endothelial cell growth medium culture media and fetal bovine serum were obtained from LONZA (Verviers, Belgium). Plastic coverslips (diameter of 25 mm) were purchased from Nunc (Thermo Fisher Scientific). PBS, collagenase, and trypsin-EDTA were obtained from Invitrogen (Eugene, OR, USA). Ficoll-Paque TM Plus was purchased from GE Healthcare (Little Chalfont, Buckinghamshire,UK).

CC1₄, Tris-HCl, thiobarbituric acid (TBA), oxidized and reduced glutathione, reduced β-nicotinamide adenine dinucleotide phosphate (NADPH), glucose-6-phosphate, 1-chloro-2,4-dinitrobenzene (CDNB), glutathione reductase, 5,5-dithio-bis-2-nitrobenzoic acid (DTNB), 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), potassium persulfate, sulfosalicylic acid (SSA), bovine serum albumin (BSA), hydrogen peroxide (H₂O₂), flavin adenine dinucleotide (FAD), 2,6-dichloroindophenol, trichloroacetic acid (TCA), Tween 20, and ethylenediaminetetraacetic acid (EDTA) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Rabbit polyclonal antibody specific for 8-hydroxy-2′-deoxyguanosine (8-8-OHdG), Rabbit polyclonal antibody specific for 4-hydroxy-2-nonenal (HNE), Rabbit polyclonal antibody specific to tumor necrosis factor alpha (TNF-α), Rabbit polyclonal antibody specific to interleukin 6 (IL-6), Rabbit polyclonal antibody specific to prostaglandin E2 (PGE2), EnVision™ + System/horseradish peroxidase (HRP), Rb (DAB+), target retrieval solution, and antibody diluent were purchased from Dako (Agilent Technologies Company, Denmark).

This assay was performed based on the glutathione recycling method by using 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) (Sigma-Aldrich) and glutathione reductase (Sigma-Aldrich). According to this method, the reaction between DTNB and GSH gave rise to the generation of 2-nitro-5-thiobenzoic acid and GSSG. Since 2-nitro-5-thiobenzoic acid was a yellow colored product, GSH concentration could be determined by measuring absorbance at 412 nm. In brief, a mixture containing 20 μL of sample and the reaction mixture consisting of 10 μL of dithiothreitol (DTT) (Sigma-Aldrich) in 6.67 mM potassium phosphate buffer (pH 7), 100 μL of sodium azide (Sigma-Aldrich) in 6.67 mM potassium phosphate buffer (pH 7), 10 μL of glutathione solution, and 100 μL of hydrogen peroxide was mixed thoroughly and incubated at room temperature for 5–10 minutes. Then, 10 μL of DTNB (5,5-dithiobis-2-nitrobenzoic acid) was added and the optical density at 412 nm was recorded at 25°C over a period of 5 min by using UV-spectrophotometer (Pharmacia LKB-Biochrom4060). Activities were expressed as nmoles/min/mg lens protein [24 (link)]. GPx activity was expressed as U/mg·protein.