Ascorbate in the ocular lens

Purpose: First, we intended to establish a method for sample preparation for measurement of ascorbate in whole rat and guinea pig lenses utilizing ultrafiltration and high performance liquid chromatography with ultraviolet radiation detection. Then, we aimed to investigate whether, in the albino rat, lens ascorbate concentration depends on solid dietary intake. Finally, we investigated if, in the pigmented guinea pig, lens ascorbate concentration may be elevated with drinking water supplementation. Background: Ascorbate is an important dietary antioxidant. Ascorbate is an essential nutrient in the human and guinea pig, while the rat is capable of synthesizing ascorbate. In vitro and in vivo studies have demonstrated the protective effect of ascorbate against cataract development in the rat and guinea pig lens. Methods: Albino Sprague Dawley rats were kept on solid diet supplemented with known amount of ascorbate for four weeks. In a second experiment, pigmented guinea pigs were kept on regular chow containing essential amount of ascorbate and drinking water supplemented with known amount of ascorbate. The animals were then sacrificed, the lenses extracted and homogenized in metaphosphoric acid. Ascorbate and other low molecular weight compounds were isolated with ultrafiltration and ascorbate was quantified with subsequent high performance liquid chromatography (HPLC) with ultraviolet radiation (UVR) detection at 254 nm. The UVR spectra for ascorbate and dehydroascorbate imply that 96% of the signal at 254 rim is ascorbate. Results: We found that external and internal calibration provided similar results. Both methods had a linear absorbance response in the range used. All rat lenses were devoid of cataract. The baseline lens ascorbate content for rats receiving no ascorbate in the diet was significantly greater than zero. Lens ascorbate concentration increased linearly with dietary ascorbate intake with a statistically significant increase rate. All guinea pig lenses were devoid of cataract. All lenses contained a detectable concentration of ascorbate. Lens ascorbate concentration increased exponentially declining with drinking water supplementation concentration, up to a saturation level. Conclusions: The method utilizing ultrafiltration and high performance liquid chromatography with ultraviolet radiation detection for measurement of whole lens ascorbate content in the rat and guinea pig lens is applicable. In the rat, lens ascorbate concentration linea