Quantification of Vitreous Ascorbate

Ascorbate concentration was quantified in triplicate, based on the ability of ascorbate to reduce Fe³⁺ to Fe²⁺ and the resulting change in the A₅₂₅ of complexes of Fe²⁺ with 2,2′-dipyridyl.25 (link) This assay was modified to permit the analysis of 15-µL samples. A freshly prepared standard curve was used for all measurements. The specificity of this assay for ascorbate in the vitreous was assessed by means of gas chromatography– mass spectrometry (GC-MS) on a subset of vitreous samples and on the ascorbate standard used for the colorimetric assay. Samples (5 µL) of vitreous humor from donor eyes or ascorbate standards were mixed with a known amount of uniformly carbon 13–labeled ascorbic acid (¹³C₆-ascorbic acid [Omicron Biochemicals, South Bend, Indiana]), dried and reacted with N,O-bis(trimethylsily)trifluoroacetamide. The sample was separated on a gas chromatograph (Varian Inc, Palo Alto, California) using a 30-m, 0.25-mm–internal diameter GC column with a 0.25-µm film (DB-5ms column; P.J. Cobert Associates Inc, St Louis, Missouri). The sample was maintained at 80°C for 1 minute, then eluted with a temperature gradient of 80°C to 300°C at 15°C/min. The injection port and transfer line were at 250°C and the source temperature at 200°C of a mass spectrometer (Finnigan MS SSQ7000; Thermo Electron Corp, Waltham, Massachusetts) operated in the electron ionization mode at 70 eV. The concentration of ascorbate in vitreous humor was calculated from the ratio of carbon 13– to carbon 12– labeled ascorbate.