Dehydro l ascorbicacid dimer

Ascorbate oxidase (AAO), 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), catalase, dehydroascorbic acid and dehydro-l-ascorbic acid dimer, o-dianisidine dihydrochloride and horseradish peroxidase type II were obtained from Sigma-Aldrich. AAO was dissolved as a stock at 1000 U ml⁻¹ in 50 mM succinate (Na⁺) buffer, pH 5.6, supplemented with 0.05% bovine serum albumin. Peroxidase was dissolved (1 μg μl⁻¹) and further diluted in the same buffer.
DKG was prepared from the commercial DHA by alkali treatment [56] . A stock of DHA (50 mM) was prepared in water (it took at least 30 min to dissolve DHA). A slight molar excess of NaOH (1.3 × ) was added and the mixture incubated at 20 °C for 6 min. Routinely, the hydrolysis was then stopped with 1 M l-tartaric acid and the pH was checked by pH paper (∼3.5–4.0). However, for samples to be fractionated by HPLC, hydrolysis was stopped with 1 M H₂SO₄ to a final pH of ∼1 or ∼6. Freshly-made DHA and DKG solutions were stored on ice before the assays.
DKG was prepared also by an iodate method [20] . A solution of ascorbic acid (0.12 M) was incubated with potassium iodate (0.36 M) for 5 min. KOH (1 M) was then added dropwise until the solution became colourless. Cold ethanol (8 vol, −20 °C) was added, and the precipitated DKG was vacuum filtered, rinsed in 70% ethanol, dried and stored at −80 °C.

Before all treatments, NEs were grown in suspension for 2 DIV and adhered to poly L-lysine-coated dishes. For the differentiation assays, L-AA (Sigma-Aldrich, St. Louis, MO, USA) was pre added to cultures at 0 (time of adhesion), 12, 24 and 48 h at a final concentration of 100 μM. PBS treatment was used as control. All-trans-retinoic acid (RA) (Sigma-Aldrich) dissolved in DMSO was added at the moment of adhesion at a final concentration of 10 μM. DMSO was used as control in these experiments. For DHA treatment, the NEs were treated with AA 100 μM at 0 and 12 h to induce differentiation. After that, dehydro-L-ascorbic acid dimer (Sigma-Aldrich, St. Louis, MO, USA) was added to cultures at 24 and 48 h at a final concentration of 100 μM. PBS was used as control. The culture medium was not changed during the treatments to allow DHA accumulation. Cell viability analysis was performed in NEs after 24, 48 and 72 h of treatment with AA 100 μM using an XTT cell proliferation kit (Biological Industries, Cromwell, CT, USA). AA concentration was measured by a FRASC colorimetric assay (#EASC-100, Bioassay system, Hayward, CA, USA), according to the manufacturer’s instructions.