Measurement of oxygen production by in vitro human and animal lenses with an oxygen electrode

Abstract

PURPOSE. This paper describes an advantageous method of measuring the activity of the enzyme catalase, which has an important antioxidative role in the lens. This method allows the measurement of catalase in whole lenses. METHODS. Exposure to UVA (99% UV-A) radiation was used to stress animal and human (Eye Bank) lenses in vitro. The ability of lens catalase to convert H 2 O 2 into O 2 was measured directly, using an oxygen electrode and meter. This method is very specific, as catalase is the only enzyme that converts H 2 O 2 to O 2. RESULTS. Catalase in the lenses of humans, rabbits, and squirrels catalyzed the production of O 2 from H 2 O 2 very efficiently. The anterior equatorial regions of these lenses were the most active O 2 producing areas. More than 95% of lens catalase activity was found in the capsule-epithelium layer. Exposure to UVA radiation, up to ~100 J/cm 2 in 18 h, strongly inhibited O 2 production from 0.77 mM H 2 O 2 by the lenses. Catalase activity decreased with increasing age. Mixed cataractous human lenses produced O2 from H 2 O 2 at only 60% of the rate of normal lenses of similar ages. Nuclear cataracts produced O 2 at only 75% of the rate of normal lenses. Alpha-tocopherol (10 -5 M) protected lens catalase activity strongly. Alpha-tocopherol is known to accumulate in and protect against cell membrane peroxidation, and against singlet oxygen formation. These oxidative mechanisms appear to contribute to catalase photoinactivation. CONCLUSIONS. The method described indicated that catalase is a crucial antioxidative enzyme in the normal lens. Its inactivation could upset the oxidation-reduction balance in the lens and stimulate lens opacification.