Glucose oxidase

dHDL was oxidized with a commercial preparation of MPO (#475911, MilliporeSigma) as described previously (28 (link)). Briefly, 25 μg of dHDL (1 mg/ml) was incubated with or without MPO-oxidase system (57 nM MPO, 100 μg/ml glucose, 20 ng/ml glucose-oxidase (49180, MilliporeSigma), and 0.05 mM NaNO₂) in total 25 μl of the activity buffer containing 10 mM sodium phosphate (pH 7.5), 200 μM diethylenetriaminepentaacetic acid, 1 mM CaCl₂ at 37 °C overnight. On the next day, half of the sample was used for Western blot analysis carried out using a monoclonal antibody specific for human PON1 and the other half used to measure PON1 lactonase activity. The samples were diluted in the lactonase assay buffer and 10 μl of 10 mM product DTNB (0.8 mM final concentration) and then 10 μl of TBBL added (1 mM final concentration). Lactonase activity was measured as change in absorbance at 405 nm, normalized to activity in absence of MPO.
Quantitation of IsoLG–Lys adducts on dHDL by IsoLG was performed as described previously (28 (link), 51 (link), 52 (link)). Briefly, dHDL with or without MPO after overnight incubation was subjected to proteolysis with Pronase and aminopeptidase M. The amount of IsoLG–lysyl–lactam was then measured by stable-isotope dilution LC/MS/MS.

β-nicotinamide adenine dinucleotide phosphate (NADP+), maleimide, glucose-6-phosphate (G6P), magnesium chloride (MgCl₂), diazonium salt, silicone oil, succinic acid, glucose, glucose oxidase, peroxidase-HRP, bilirubin, dyphylline, excipients, surfactants, preservatives and stabilizers were procured from MilliporeSigma (St. Louis, MO, USA). Bromocresol Green (BCG) was obtained from Affymetrix (Santa Clara, CA, USA). Creatinine kits (2-step colorimetric readout) were purchased from Abcam (Cambridge, UK). Urine calibrator samples were procured from CST Technologies (Great Neck, NY, USA).

PCL (average molecular mass: 80,000 g/mol), chloroform (purity > 99%), ethanol (99.5%), glucose oxidase (from Aspergillus niger, 145,200 units/g solid), horseradish peroxidase (297,000 units/g solid, suitable for manufacturing of diagnostic kits and reagents), trehalose dihydrate (from Saccharomyces cerevisiae, > 99%,) and 3,3’,5’,5’-tetramethylbenzidine (purity > 99%) (TMB) were purchased from Merck KgaA, Corp. (Tokyo, Japan). D (+)-Glucose was purchased from Hayashi Pure Chemical Ind., Ltd. (Osaka, Japan). A phosphate buffer solution was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). A high-voltage power supply (model HJPQ 30P1, Matsusada Precision, Japan) was used. The syringe pump was purchased from KD Scientific, Inc. (Holliston, MA) (model KDS 200).
A syringe of 5 mL with a metallic blunt tip of 18 gauge was used. Air plasma treatment was performed using a plasma generator (Sakigake YHS-R, Japan), and the water contact angle was measured using a solid-liquid interface analyser DropMaster 300 (Kyowa Interface Science Co., Ltd., Japan).

Sections were washed 3 times in 1× TBS and the endogenous peroxidase activity was blocked with 0.3% H₂O₂. Blocking buffer contained 2% bovine serum albumin and 0.3% Triton X-100 (both purchased from Merck KGaA). Then sections were incubated overnight at RT with polyclonal chicken anti-β-galactosidase (ab9361, RRID: AB_307210, Abcam, Cambridge, UK), diluted 1:4000 in blocking buffer. After TBS washes, sections were incubated for 2 h at RT with peroxidase conjugated donkey anti-chicken IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA), diluted 1:500 in blocking buffer. Visualization were performed using nickel(II) sulfate hexahydrate/3,3′-diaminobenzidine tetrahydrochloride (Merck KGaA) as chromogen and glucose oxidase (Merck KGaA) [49 (link)]. Sections were mounted onto gelatinized slides, allowed to dry overnight, transferred into ascending ethanol solutions (50%, 70%, 96%, absolute for 5 min, respectively), then cover slipped with PERTEX mounting medium. Brightfield images of PrL, BMA, CeC, BLA, layer V of the primary S1, CA1 and MHb, according to Paxinos and Franklin [47 ] were acquired using a Nikon Microphot-FXA microscope (Nikon, Tokyo, Japan), then contrasted using Photoshop CS6 (Adobe, San José, CA, USA).