Ascorbic Acid as a Free Radical Scavenger in Porcine and Bovine Aqueous Humour

Aim: To study the antioxidant activity, UV absorption, concentration and stability of ascorbic acid (AA) in porcine and bovine aqueous humour (AH). Methods: Porcine and bovine AH was taken within 5 min after death and frozen at –70°C. The characteristic UV absorption band of AA and the concentration of AA in AH was determined by UV spectrophotometry. The antioxidant activity of AA to serve as a free radical scavenger in AH has been determined by using a novel fluorescent probe for antioxidants, the azoalkane 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO). The fluorescence lifetime and intensity of this probe reflect the concentration of dissolved antioxidants. The time-resolved fluorescence of DBO (laser excitation at 351 nm) in AH and in a neutral phosphate-buffered saline (PBS) solution containing only the natural amount of AA as an additive were measured. Results: The characteristic UV absorption band of AA has its maximum at 266 nm in AH. The concentration of AA in porcine and bovine AH was found to be 0.547 ± 0.044 and 1.09 ± 0.16 mM, respectively, by spectrophotometry. The fluorescence lifetime of the probe DBO was reduced from 320 ± 5 ns in pure aerated PBS to 205 ± 5 ns in porcine AH and 165 ± 3 ns in bovine AH. A detailed kinetic analysis of the lifetime shortening suggests that AA contributes approximately 75 and 85% to the antioxidant activity of porcine and bovine AH, respectively. Conclusions: Our experiments suggest that AA is the major contributor to the antioxidant activity of porcine and bovine AH. The role of AA to serve as an antioxidant in AH is discussed. In addition, UV spectrophotometry is established as an alternative method to determine the concentration of AA in AH.