Lysosomal enzyme leakage during the hypoxanthine/xanthine oxidase reaction

Impairment of lysosomal stability due to reactive oxygen species generated during the oxidation of hypoxanthine by xanthine oxidase was studied in rat liver lysosomes isolated in a discontinuous Nycodenz gradient. Production of O ₂ ^⋅ and H₂O₂ during the hypoxanthine/xanthine oxidase reaction occurred for at least 5 min, while lysosomal damage, indicated by the release of N-acetyl-β-glucosaminidase, occurred within 30 s, there being no further damage to these organelles thereafter. The extent of lysosomal enzyme release increased with increasing xanthine oxidase concentration. Superoxide dismutase and catalase did not prevent lysosomal damage during the hypoxanthine/xanthine oxidase reaction. Lysosomes reduced xanthine oxidase activity, as assessed in terms of O₂ consumption, only slightly but substantially inhibited in a competitive manner the O ₂ ^⋅ -mediated reduction of cytochrome c. This inhibition was almost completely reversed by potassium cyanide, thus pointing to the presence of a cyanide-sensitive Superoxide dismutase in the lysosomal fraction. However, potassium cyanide did not affect the hypoxanthine/xanthine oxidase-mediated lysosomal damage, thus suggesting an inability of the lysosomal superoxide dismutase to protect the organelles. Negligible malondialdehyde formation was observed in the lysosomes either during the hypoxanthine/xanthine oxidase reaction or with different selective experimental approaches known to produce lipid peroxidation in other organelles such as microsomes and mitochondria. These results are interpreted in terms of a possible lysosomal membrane permeability to O ₂ ^⋅ causing organelle impairment by a process that, though leading to enzyme-marker leakage, does not involve lipid peroxidation.